CN108315281A - One bacillus subtilis and its application in preventing Red Spot Disease of Peony Tree - Google Patents
One bacillus subtilis and its application in preventing Red Spot Disease of Peony Tree Download PDFInfo
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- CN108315281A CN108315281A CN201810343840.6A CN201810343840A CN108315281A CN 108315281 A CN108315281 A CN 108315281A CN 201810343840 A CN201810343840 A CN 201810343840A CN 108315281 A CN108315281 A CN 108315281A
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- bacillus subtilis
- peony
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- spot disease
- tree
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
Application the invention discloses a bacillus subtilis and its in preventing Red Spot Disease of Peony Tree.The bacterial strain is bacillus subtilis (Bacillus subtilis) bacterial strain HZ08 3, it detaches and obtains from the wetland river silt of Heze, it is preserved within 26th China Committee for Culture Collection of Microorganisms's common micro-organisms center in September in 2017, preserving number is CGMCC No.14679.Test result shows：3 biological control agents of bacillus subtilis strain HZ08 prevent Red Spot Disease of Peony Tree significant effect, and control effect reaches 75.95%, and tree peony grows fine, and no poisoning generates.
Description
Technical field
The invention belongs to technical field of plant disease biological control.Specifically, the present invention relates to one plant of bacillus subtilis
Bacterium and its application in preventing Red Spot Disease of Peony Tree, and the biological control agent using bacillus preparation.
Background technology
Tree peony is one of China's tradition famous flower and world-renowned flowers, is described as colored king, national flower by compatriots since ancient times
Deng being described as middle national flower by countries in the world.In recent years, as the rapid extension of Peony growing area, kind increase, Peony disease
Occurrence injury is in rising trend, and wherein Red Spot Disease of Peony Tree (Cladosporium paeoniae Pass.) is that endanger tree peony most tight
One of the disease of weight, which also endangers Chinese herbaceous peony.Red Spot Disease of Peony Tree is by mould (the Cladosporium paeoniae of tree peony branch spore
Pass.) cause, the leaf of main harm tree peony and green stem, it is this that the scab on face of blade and stem keeps dark violet red for a long time
The cardinal symptom feature of disease, mid-June to late July are disease morbidity peak period.After pathogen infection tree peony, tree peony pattern is caused
Decline, growth is impaired and the root bark of tree peony is lacking both in quantity and quality, causes flower-grower's economic loss heavy, and the outburst of disease seriously constrains me with popular
The development of state's tree peony industrialization.
At present frequently with Agro-chemicals control Red Spot Disease of Peony Tree in production.Chemical prevention easily causes pesticide residue, pollution
Environment, and the long-time service of chemical pesticide so that pathogen develops immunity to drugs to it, causes control effect to decline and even loses
It loses.
Biological control is that the generation of disease is reduced using the antagonism between species, to safety of human and livestock, environment compatibility
Good and pathogen is not likely to produce drug resistance, thus the advantage not having with chemical bactericide, is just gradually becoming and is being killed instead of chemistry
The best mode of microbial inoculum.And obtain the basis that efficient Antagonistic Fungi is biological control.Bacillus subtilis (Bacillus
Subtilis) it is the strain being reported earliest for controlling plant diseases.
Bacillus is the general name of a kind of aerobic or amphimicrobian production gemma Gram-positive bacillus, and physiological characteristic is rich
It is rich various, it is widespread in nature, is the important microbe population of soil and plant surface rhizosphere, to the nontoxic nothing of people and animals
Evil, it is free from environmental pollution, a variety of antibiotic and enzyme can be generated, there is broad spectrum antibiotic activity.Bacillus feature outstanding is to produce
Sheng Neisheng is heat-resisting and has the gemma of very strong resistance to ultraviolet light, electromagnetic radiation and some chemicals, this is conducive to give birth to
The production of anti-microbial inoculum and formulation are also conducive to its survival in the environment, colonize and breed.Due to the bacillus speed of growth
Soon, nutritional need is simple, can form the stronger gemma of resistance, and more non-bacillus has effective viable bacteria in product development
The advantages such as quantity is high, performance is stablized and attract attention.
Invention content
The object of the present invention is to provide a kind of new microorganism --- withered grass gemma for efficiently preventing Red Spot Disease of Peony Tree
Bacillus strain HZ08-3.The present invention bacillus subtilis strain have the speed of growth is fast, yield is big, resistance is strong, in plant
(especially tree peony) surface can rapid, high volume the features such as colonizing, therefore have a good application prospect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：Bacterial strain provided by the present invention is withered grass gemma
Bacillus (Bacillus subtilis) bacterial strain HZ08-3, detaches from the wetland river silt of Heze and obtains, in September, 2017
It is preserved within 26th China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Court of Beijing
The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica), preserving number is CGMCC No.14679.It has with
Lower biological characteristics：Colonial morphology is circle in beef extract-peptone agar medium or LB (Luria-Bertani) culture medium
Shape or irregular shape, newborn white partial yellow, rough surface play fold and are cultivated at 28 DEG C in beef extract-peptone agar medium
Two days later, microscopy, somatic cells are in rod-shaped, can be moved.It is positive (being control with Escherichia coli) through Gram's staining.Through gemma
Observe that gemma is oval or column after dyeing.
The cultural method or propagation method of bacillus subtilis (B.subtilis) bacterial strain HZ08-3 of the present invention include：
(1) Nostoc commune Vanch, which preserves, uses LB culture mediums, is formulated and is：Tryptone 10g, yeast extract 5g, sodium chloride
10g, 15~20g of agar, distilled water 1000mL, pH are adjusted to 7.1~7.3；
(2) laboratory fluids culture uses LB liquid medium, is formulated and is：Tryptone 10g, yeast extract 5g, chlorine
Change sodium 10g, distilled water 1000mL, pH are adjusted to 7.1~7.3；
(3) solid culture based formulas：Gu the mass ratio of material and inorganic salts, the solid material and inorganic salts is 98.6: 0.4；Institute
It states solid material and is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=50: 25: 20: 5；The component and weight of the inorganic salts
Than for：Potassium dihydrogen phosphate 18%, magnesium sulfate 5%, ammonium sulfate 12%, precipitated calcium carbonate 65%.
(4) bulk fermentation culture formula：With solid culture based formulas in (3).
The present invention also provides a kind of biological control agent for preventing Red Spot Disease of Peony Tree, the biological control agent includes
Bacillus subtilis (B.subtilis) the bacterial strain HZ08-3.
Also, the present invention also provides a kind of biological control method for preventing Red Spot Disease of Peony Tree, the biological controls
Method include to the peony plant with red spot disease apply above-mentioned bacillus subtilis (B.subtilis) bacterial strain HZ08-3 or
Above-mentioned biological control agent.
It is male in prevention that present invention provides above-mentioned bacillus subtilis strain HZ08-3 or above-mentioned biological control agent
Purposes in red red spot disease.
The preparation method of biological control agent of the present invention includes the following steps：
(1) seed liquor of the bacillus subtilis strain HZ08-3 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 26~28 DEG C；
(3) sterile water is added in the culture of step (2) culture to mix, filtrate is seeded to bulk fermentation culture by filtering
Base, 26~28 DEG C of room temperature, 85% or more relative humidity fermenting cellar in carry out fermented and cultured.
In one embodiment of the invention, the preparation method comprises the following steps：
(1) spore of the bacillus subtilis strain HZ08-3 is transplanted to LB liquid medium, 26~28 DEG C of shaking tables
3~5d of shaken cultivation obtains seed liquor；
(2) ratio of seed liquor in mass ratio 10% prepared by step (1) is inoculated into solid medium, 26~28 DEG C
3~5d of lower shaken cultivation；
(3) ratio of the culture sterile water in mass ratio 1: 15 of step (2) culture is mixed, sterile gauze mistake is used in combination
Filter, by filtrate by volume 1: 6 ratio be seeded to fermentation medium, 26~28 DEG C of room temperature, 85% or more relative humidity hair
8~9d of fermented and cultured in ferment room.
Wherein, the LB liquid medium in step (1) is formulated as tryptone 10g, yeast extract 5g, sodium chloride
10g, distilled water 1000mL, pH are adjusted to 7.1~7.3.
Solid medium in step (2) includes solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.6:
0.4；The solid material is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=50: 25: 20: 5；The component of the inorganic salts
And weight ratio is：Potassium dihydrogen phosphate 18%, magnesium sulfate 5%, ammonium sulfate 12%, precipitated calcium carbonate 65%.
The solid medium of the same step of fermentation medium (2) in step (3).
Experiment show bacillus subtilis strain of the invention have the speed of growth is fast, yield is big, resistance is strong, planting
Object (especially tree peony) surface can rapid, high volume the features such as colonizing, therefore have a good application prospect.By the withered grass gemma
Biological control agent prepared by bacillus can not only efficiently prevent Red Spot Disease of Peony Tree evil, moreover it is possible to effectively facilitate tree peony growth, be
A kind of biological control agent of great application prospect.The microorganism formulation can be used as biological pesticide or bio-feritlizer, prevention male
Red red spot disease.Test result shows：The prevention of bacillus subtilis (B.subtilis) bacterial strain HZ08-3 solid fermentation cultures is male
Red red spot disease significant effect, control effect reaches 75.95%, and tree peony grows fine, and no poisoning generates.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are merely exemplary, and it is not intended to limit the scope of the present invention in any way.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are within the scope of the invention.
Embodiment 1
1, the isolation and purification of bacillus subtilis (B.subtilis) HZ08-3
Bacillus subtilis (B.subtilis) HZ08-3 of the present invention is that dilution-plate method and tablet are used from river silt
Method of scoring separation obtains, and separation method is：
(1) separation of bacillus
River silt sample was derived from Heze wetland on May 21st, 2013.1g river silt is weighed in 100mL sterile waters, is placed in 30
150rpm shakes 10min in DEG C shaking table, is subsequently placed in 60 DEG C of water-baths and is incubated 30min, takes 100 μ L 10-2、10-3、10-4It is dilute
It releases liquid to be coated on LB culture medium flat plates, each gradient coating three is parallel, at 30 DEG C after culture 2d on picking LB culture mediums
The microbial bacteria of different shape falls within and crosses on LB culture medium flat plates, and bacterium colony growing state is observed in timing.Then using flat
Plate method of scoring purifies Bacillus strain, and number preserves respectively.
(2) screening of the efficient antagonism bacillus subtilis of Red Spot Disease of Peony Tree
1. primary dcreening operation：Using opposite culture method, PDA (Potato Dextrose Agar) tablet is prepared, with card punch in bud
Spore bacillus, Red Spot Disease of Peony Tree pathogen edge take the bacteria cake of a diameter of 5mm, be implanted in respectively tablet it is opposite both sides center, 25
DEG C constant temperature incubation observes inhibiting effect of the bacillus subtilis to pathogen day by day.
2. secondary screening：The bacillus subtilis strain with efficient antagonistic activity screened is subjected to secondary screening, is mainly passed through
Cross temperature tolerance, resistance to acid and alkali, drug-resistant test, screen the preferable Bacillus strain of patience, carry out potting controlling experiment and
Field trial, and the bacterial strain HZ08-3 obtained to screening is identified.
The present inventor obtains one plant of bacillus subtilis that can efficiently prevent Red Spot Disease of Peony Tree by a large amount of screening operations
(B.subtilis)HZ08-3.It is demonstrated experimentally that the bacillus subtilis solid fermentation culture is shown in preventing Red Spot Disease of Peony Tree
Very efficient control effect is shown so that tree peony grows fine.Thus, bacillus subtilis of the invention is that have to answer extensively
With the bacillus subtilis new strains of foreground, it can be used for preparing the biological control agent of prevention Red Spot Disease of Peony Tree disease.
2, bacterial strain is identified
(1) Microbiological Characteristics：In beef extract-peptone agar medium or LB culture mediums colonial morphology be it is round or
Irregular shape, newborn white partial yellow, rough surface play fold；In beef extract-peptone agar medium, cultivated two days at 28 DEG C
Afterwards, microscopy, somatic cells are in rod-shaped, can be moved.It is positive (being control with Escherichia coli) through Gram's staining.Through spore staining
Gemma is oval or column for observation afterwards.
(2) molecular biological characteristic
The 16s rDNA gene sequencings result of the bacterial strain is following (SEQ-1)：
GGTTACCTTACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC
ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAG
AACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCA
GGGCCAAAGGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCC
CAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGA
CGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGAC
CTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGA
GTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCT
AACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTC
AGCGTCAGTTACAGACCAGAGAGTCGCTTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGT
GGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCAC
ATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGC
GGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTT
CTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCAT
TGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTTTC
CAGTTGGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAG
TGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAG
TTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATC
TGTCCGT。
Bacterial strain HZ08-3 is accredited as bacillus subtilis (Bacillus subtilis), is protected within 26th in September in 2017
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.14679.
Embodiment 2
1, Bacillus fermentation process
LB liquid medium formula is：Tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH
It is adjusted to 7.1~7.3.
Solid medium is made of solid material, inorganic salts and water, and the mass ratio of the solid material and inorganic salts is 98.6: 0.4；Institute
It states solid material and is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=50: 25: 20: 5；The component and weight of the inorganic salts
Than for：Potassium dihydrogen phosphate 18%, magnesium sulfate 5%, ammonium sulfate 12%, precipitated calcium carbonate 65%.Preparation method：Suitable water is added
(10-15 times of quality) dissolves inorganic salts, obtains inorganic salt solution；Inorganic salt solution is mixed with solid material, and water final adjustment is added
The water content of solid medium is 40-60%, and solid medium is held in the hand, has water in webs but not drippage is advisable.
The same solid medium of bulk fermentation culture medium.
The a large amount of solid fermentation process of bacillus subtilis (B.subtilis) bacterial strain HZ08-3：
1. strain seed liquor culture
By a small amount of spore of bacillus subtilis strain (B.subtilis) HZ08-3 pickings from test tube slant, LB liquid is moved to
In body culture medium, 27 DEG C of shaking table 3~5d of shaken cultivation, this is seed liquor；
2. the culture of solids manufacture strain
Seed liquor is inoculated into 10% ratio in solid medium (500mL triangular flasks), 27 DEG C of constant temperature incubations 3~
5d, intermediate multiple oscillation；
3. a large amount of solid fermentations：
2. the culture sterile water of middle solid culture is pressed into 1: 15 dilution proportion, is used in combination sterile gauze to filter, removes thick
Slag, as produces bacterium solution, and inoculative proportion is inoculated in bulk fermentation culture medium by 1: 6.The raw material of inoculation is placed in (27 DEG C of fermenting cellar
With 85% or more relative humidity) in 8~9d of fermented and cultured, active bacteria can reach 3,200,000,000/gram.
It is it in order to which clear bacillus subtilis (B.subtilis) bacterial strain HZ08-3 is to the control effect of Red Spot Disease of Peony Tree
It promotes and applies and foundation is provided.We test in March, 2016~July in Heze city of Shandong province, now report test result
It is as follows：1 material to be tested and method
1.1 reagent agent
By bacillus subtilis (B.subtilis) bacterial strain HZ08-3 solids hair of 2 fermentation process of embodiment of the present invention production
Ferment culture (3,200,000,000/g)；(Xinyi City of Jiangsu Province Zhong Gang pesticide chemicals Co., Ltd produces 50% carbendazol wettable powder, city
It sells).
1.2 test materials and controlling object
Test material is tree peony；Controlling object is Red Spot Disease of Peony Tree (Cladosporium paeoniae Pass.).
1.3 experimental field situations
Experimental field it is located at Heze city of Shandong province flood mausoleum tree peony base.In Heze Prefecture, Red Spot Disease of Peony Tree occurring degree Ying Yin
Kind and plot it is different and different, have up to 80% or more peony garden disease incidence 50% or so, seriously ill garden incidence
Even 90% or more.Experiment soil types is moisture soil, and the content of organic matter 3.63%, pH value 7.0, all experimental plots plant
Training condition and management measure are consistent, and Red Spot Disease of Peony Tree is in early stage when dispenser.
1.4 experimental designs and arrangement
This experiment totally 3 processing, respectively bacillus subtilis (B.subtilis) bacterial strain HZ08-3 solid fermentation cultures
15 times of liquid of object, 50% benomyl wettable powder, 600 times of liquid, not dispenser clear water compare, and each handle 4 repetitions, each to locate
60 plants of reason.On March 11st, 2016, May 16, June 15 and July 10, respectively sprinkling is primary, and spray instrument is workers and peasants -16
Type sprayer.
1.5 pilot surveies and computational methods
1.5.1 meteorological condition
1st dispenser (March 11) same day is fine, 3 grades of wind-force, and the highest temperature is 12 DEG C, and the lowest temperature is 2 DEG C, relatively wet
Degree is 80%.2nd dispenser (May 16), the same day is fine, 3 grades of wind-force, and the highest temperature is 28 DEG C, and the lowest temperature is 15 DEG C, relatively
Humidity is 75%；3rd dispenser (June 15) same day is cloudy, 4 grades of wind-force, and the highest temperature is 28 DEG C, and the lowest temperature is 18 DEG C,
Relative humidity is 80%；The 4th dispenser (July 10) same day is cloudy, 3 grades of wind-force, and the highest temperature is 32 DEG C, the lowest temperature 23
DEG C, relative humidity 80%.
1.5.2 drug effect and security survey
Efficacy survey：14d is investigated after last time dispenser.Each 50 plants of processing investigation, investigation plant basal part of stem and
Blade, Investigate incidence record disease index and calculate disease index.
Security survey：The safety of 7d and 14d observations tree peony, occurs if any poisoning, retouches in detail after first time dispenser
It states symptom of chemical damage and determines poisoning degree by poisoning grading standard.
Red Spot Disease of Peony Tree grade scale：
0 grade：Complete stool stem, blade health, it is disease-free.
1 grade：Amount of speckle is at 3 hereinafter, top stem has fragmentary dot within the scope of basal part of stem 10cm, and complete stool 1/4 is with inferior lobe
Piece is fallen ill, and the scab number on average each compound leaf is at 2 hereinafter, scab is not linked to be piece.
2 grades：3~5 spots within the scope of basal part of stem 10cm, top stem spot is sparse, the morbidity of 1/4~1/2 blade, average every
There are 3~5 scabs on a compound leaf.
3 grades：There are 6~10 spots within the scope of basal part of stem 10cm, top stem spot is closeer, and the morbidity of 1/2~3/4 blade is put down
There are 6~10 scabs on each compound leaf, spot is intensive, and 1/3 is dried-up with blade.
4 grades：There are 10 or more spots within the scope of basal part of stem 10cm, top stem spot is intensive, and 3/4 is fallen ill with blade, puts down
There are 10 or more scabs on each compound leaf, spot is intensive and connected in blocks, and 1/2 is dried-up with blade.
1.5.3 drug effect computational methods
Control effect is calculated by formula (1), (2)：
In formula：CK0--- disease index before blank district dispenser；
CK1--- disease index after blank district dispenser；
PT0————Disease index before dispenser before chemicals treatment；
PT1————Disease index after dispenser after chemicals treatment.
1.5.4 tree peony is directly affected
Observing medicament, whether there is or not poisoning to tree peony, record the type and extent of poisoning.In addition, also to record to tree peony growing way
It influences.
Poisoning is recorded with following manner：
If (a) poisoning can be measured or calculate, absolute value representation, such as plant height, setting percentage are used.
(b) in other cases, it can estimate poisoning degree and frequency by following two methods：
1. the poisoning degree per cell is recorded according to poisoning stage division, with-,+, ++, +++, ++++it indicates.
Poisoning stage division：
—：Without poisoning；
+：Slight poisoning, does not influence plant growth；
++：Moderate poisoning, it is resilient, phytomass will not be reduced；
+++：Moderate poisoning, influences plant normal growth, and a degree of reduction is caused to phytomass；
++++：Serious poisoning, plant growth are obstructed, and phytomass reduces serious.
2. by chemicals treatment district compared with blank district, its poisoning percentage is evaluated.Meanwhile wanting accurate description plant
Symptom of chemical damage (dwarfing, chlorisis, deformity etc.).
2 results
Control effect of 2.1 reagent agents to Red Spot Disease of Peony Tree
Reagent agent is to the control effect of Red Spot Disease of Peony Tree as shown in table 1-2.Table 2 is the results show that bacillus subtilis
(B.subtilis) bacterial strain HZ08-3 solid fermentation cultures and 50% carbendazol wettable powder all have Red Spot Disease of Peony Tree
Preferable control effect, wherein the former control effect reach 75% or more.
From the point of view of the disease index of Red Spot Disease of Peony Tree field trial (table 2), 14d after last time dispenser, bacillus subtilis
Bacterium (B.subtilis) HZ08-3 control effects are 75.95%, hence it is evident that anti-higher than 50% carbendazol wettable powder 58.27%
Control effect.
Red Spot Disease of Peony Tree situation before 1 three processing of table
2 three processing of table are to Red Spot Disease of Peony Tree control effect
2.2 tree peony security surveys
It is observed through dispenser 7d and 14d, each chemicals treatment district is compared with check plot, and tree peony growth is normal, and no poisoning generates, and says
Bright bacillus subtilis solid fermentation culture is for examination concentration to tree peony safety.
3 brief summaries
3.1 from the point of view of disease index and control effect, bacillus subtilis (B.subtilis) bacterial strain HZ08-3 (preserving numbers
CGMCC No.14679) there is preferable control effect to Red Spot Disease of Peony Tree, 14d control effects are up to 75% after last time dispenser
More than.
3.2 in terms of tree peony growing way, and bacillus subtilis (B.subtilis) bacterial strain HZ08-3 has growth-promoting long-acting tree peony
Fruit, without poisoning, securely and reliably.
SEQUENCE LISTING
<110>Shandong Forest Science Academy
<120>One bacillus subtilis and its application in preventing Red Spot Disease of Peony Tree
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1393
<212> DNA
<213>Bacillus subtilis（Bacillus subtilis）The 16s rDNA gene orders of bacterial strain HZ08-3
<400> 1
ggttacctta ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg gtgtgtacaa 60
ggcccgggaa cgtattcacc gcggcatgct gatccgcgat tactagcgat tccagcttca 120
cgcagtcgag ttgcagactg cgatccgaac tgagaacaga tttgtgggat tggcttaacc 180
tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg tgtgtagccc agggccaaag 240
ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtcacctt 300
agagtgccca actgaatgct ggcaactaag atcaagggtt gcgctcgttg cgggacttaa 360
cccaacatct cacgacacga gctgacgaca accatgcacc acctgtcact ctgcccccga 420
aggggacgtc ctatctctag gattgtcaga ggatgtcaag acctggtaag gttcttcgcg 480
ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag 540
tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa tgcgttagct gcagcactaa 600
ggggcggaaa ccccctaaca cttagcactc atcgtttacg gcgtggacta ccagggtatc 660
taatcctgtt cgctccccac gctttcgctc ctcagcgtca gttacagacc agagagtcgc 720
tttcgccact ggtgttcctc cacatctcta cgcatttcac cgctacacgt ggaattccac 780
tctcctcttc tgcactcaag ttccccagtt tccaatgacc ctccccggtt gagccggggg 840
ctttcacatc agacttaaga aaccgcctgc gagcccttta cgcccaataa ttccggacaa 900
cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctggtt 960
aggtaccgtc aaggtgccgc cctatttgaa cggcacttgt tcttccctaa caacagagct 1020
ttacgatccg aaaaccttca tcactcacgc ggcgttgctc cgtcagactt tcgtccattg 1080
cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1140
gccgatcacc ctttccagtt gggctacgca tcgtcgcctt ggtgagccgt tacctcacca 1200
actagctaat gcgccgcggg tccatctgta agtggtagcc gaagccacct tttatgtctg 1260
aaccatgcgg ttcagacaac catccggtat tagccccggt ttcccggagt tatcccagtc 1320
ttacaggcag gttacccacg tgttactcac ccgtccgccg ctaacatcag ggagcaagct 1380
cccatctgtc cgt 1393
Claims (8)
1. the deposit number of bacillus subtilis (Bacillus subtilis) bacterial strain HZ08-3, the bacterial strain are CGMCC
No.14679。
2. purposes of the bacillus subtilis strain HZ08-3 described in claim 1 in preventing Red Spot Disease of Peony Tree.
3. a kind of biological control agent, including bacillus subtilis strain HZ08-3 described in claim 1.
4. the preparation method of the biological control agent described in claim 3, characterized in that include the following steps：
(1) seed liquor of bacillus subtilis strain HZ08-3 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 26~28 DEG C；
(3) sterile water being added in the culture of step (2) culture to mix, filtrate is seeded to bulk fermentation culture medium by filtering,
26~28 DEG C of room temperature, 85% or more relative humidity fermenting cellar in carry out fermented and cultured.
5. the preparation method of biological control agent as claimed in claim 4, characterized in that
Solid medium in the step (2) includes solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.6:
0.4；The solid material is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=50: 25: 20: 5；The component of the inorganic salts
And weight ratio is：Potassium dihydrogen phosphate 18%, magnesium sulfate 5%, ammonium sulfate 12%, precipitated calcium carbonate 65%；
The solid medium of the same step of fermentation medium (2) in the step (3).
6. the preparation method of biological control agent as claimed in claim 5, characterized in that include the following steps：
(1) spore of the bacillus subtilis strain HZ08-3 is transplanted to LB liquid medium, 26~28 DEG C of shaking table oscillations
3~5d of culture obtains seed liquor；
(2) ratio of seed liquor in mass ratio 10% prepared by step (1) is inoculated into solid medium, is shaken at 26~28 DEG C
Swing 3~5d of culture；
(3) ratio that the culture of step (2) culture is added to sterile water in mass ratio 1: 15 mixes, and sterile gauze mistake is used in combination
Filter, by filtrate by volume 1: 6 ratio be seeded to bulk fermentation culture medium, 26~28 DEG C of room temperature, 85% or more relative humidity
Fermenting cellar in 8~9d of fermented and cultured.
7. purposes of the biological control agent in preventing Red Spot Disease of Peony Tree described in claim 3.
8. a kind of biological control method for preventing Red Spot Disease of Peony Tree, characterized in that applied to the peony plant with red spot disease
With the biological control agent described in bacillus subtilis strain HZ08-3 described in claim 1 or claim 3.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130958A (en) * | 2014-07-24 | 2014-11-05 | 山东省林业科学研究院 | Bacillus subtilis and application of bacillus subtilis in preventing and curing peony root-knot nematode |
| CN105053015A (en) * | 2015-07-15 | 2015-11-18 | 河南科技大学 | Biopesticide for controlling cercospora varrireolorWinter, and preparation method for biopesticide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130958A (en) * | 2014-07-24 | 2014-11-05 | 山东省林业科学研究院 | Bacillus subtilis and application of bacillus subtilis in preventing and curing peony root-knot nematode |
| CN105053015A (en) * | 2015-07-15 | 2015-11-18 | 河南科技大学 | Biopesticide for controlling cercospora varrireolorWinter, and preparation method for biopesticide |
Non-Patent Citations (1)
| Title |
|---|
| SONG, P ET AL: "OPTIMIZATION OF FERMENTATION CONDITIONS OF BIOCONTROL STRAIN BACILLUS SUBTILIS HS5B5", 《PROCEEDINGS OF THE 2016 INTERNATIONAL CONFERENCE ON BIOTECHNOLOGY & MEDICAL SCIENCE》 * |
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