CN106967635A - A kind of preparation method for the biocontrol agent for preventing and treating apple tree fungal disease - Google Patents
A kind of preparation method for the biocontrol agent for preventing and treating apple tree fungal disease Download PDFInfo
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Abstract
The invention discloses a kind of preparation method for the biocontrol agent for preventing and treating apple tree fungal disease, it is related to bacteria agent technical field, the specially preparation method of cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN deposit numbers are CGMCC NO.13080, the microbial inoculum has significant prevention effect for preventing and treating apple anthrax leaf blight, alternaria leaf spot of apple, and the microbial inoculum prevents and treats function for common apple tree leaf diseases and other Disease of Apple Trees.The biocontrol agent can not only substitute chemical prevention and control method completely, exclude food security hidden danger, and bacterium obtains prominent effect in terms of the validity and antibacterial broad spectrum activity of the preventing and treating common leaf diseases of apple tree.At the same time the drappus microbial inoculum possesses the characteristics such as high, the strong, broad-spectrum antibacterial of heat resistance of fermentative activity, can also prevent and treat other fruits and vegetables such as Common Diseases such as cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato.
Description
Technical field
The present invention relates to bacteria agent technical field, and in particular to a kind of biocontrol agent of preventing and treating apple tree fungal disease
Preparation method.
Background technology
In the growth course of apple tree, common apple tree fungal disease mainly has apple anthrax leaf blight, apple spot
Select defoliation, canker of apple fruit, apple phytophthora rot etc..Wherein, apple anthrax leaf blight (Glomerella leaf spot) is
A kind of new expression popular in the last few years, the Cao Keqiang professors of pest and disease damage prevention and control research department and its seminar find, in China
Every July has orchard worker to react the generation of doubtful apple anthrax leaf blight and endanger serious successively since 2009.The disease is main
It was found that in the kind such as loud, high-pitched sound drawing, Jin Guan, Qin Guan and Qiao Najin.Qingdao Agricultural University Li Baohua teaches seminar and germ is carried out
Separation, culture and identification, it is the referred to as anthrax leaf spoting bacteria (Glomerella as caused by anthrax-bacilus to determine the disease
cingulata).The disease belongs to apple tree leaf portion fungal disease, and the dun subcircular disease in edge occurs in disease blade at initial stage
Spot, can finally cause blade blackening, in shrivelled shape when meeting hot and humid environment, then come off.Fruit can also infect this disease, fruit
Upper formation subcircular necrotic plaque.
The sick preventing and treating mainly spray before First aggression with chemical pesticide at present based on protection, biological preventing control method
There is the endogeny rayungus A-1 for retrieving Zhu Xueming researchs to can induce resistance of the apple tree to anthrax leaf blight, preventive effect reaches
70.42%.Because the disease just comes into vogue recent years in China, thus it is also immature to the sick Prevention Technique, be currently
Rely mainly on prevention, therapeutic effect is bad.
Such as article " control effect of several medicaments to apple anthrax leaf blight ", Song Liangdong, Wang Xuelian, bean powder is graceful, what
Gorgeous dimension, Shaanxi agricultural sciences 2016,62 (11):46-48, discloses experiment and has selected 5 kinds of medicaments, is carried out in Gala apple garden
The prevention and control of apple anthrax leaf blight, as a result show：25% Amici can be produced up to 1000 times of liquid of (Fluoxastrobin) suspending agent to apple tree
Crude drug does harm to, and should not be used in Gala apple garden；78% bohr MnZn (section wins) 400 times of liquid of wettable powder and 80% maneb
Zinc (big raw) 500 times of liquid of wettable powder, it is not only preferable to apple anthrax leaf blight preventive effect, and also have to other several defoliations
Simultaneous anti-effect, can be widely applied well.
Such as article " prevention effect of the different bactericide to apple anthrax leaf blight ", Wang Bing, Wang Caixia, Shi Xiangpeng, etc.,
Plant protection, 2014 (6):176-180, discloses to filter out effective bactericide of preventing and treating apple anthrax leaf blight, uses
It is outdoor be first inoculated with after the method that is inoculated with after dispenser and first dispenser, test the interior suction therapeutic effect of 6 kinds of medicaments and the guarantor of 8 kinds of medicaments
Protect effect.Used in 72h after infection process in pyraclostrobin, or 24h after infection process using Prochloraz to disease
The aobvious disease of spot has certain therapeutic effect.Bordeaux mixture 18d after spraying, still reaches to the inhibition that anthrax leaf spoting bacteria infects
50%, oxime bacterium Tebuconazole, 3 kinds of medicaments of alkene acyl pyrazoles ester and azoles ether Carbatene after administration the 11st day, it protects effect
Fruit is with compare still that there were significant differences, and the lasting period reaches 11d, and 4 kinds of Mancozeb, thiophanate-methyl, Kocide SD and Prochloraz are protected
The lasting period of agent can only maintain 6d.Therefore the preventing and treating of anthrax leaf blight should be based on Bordeaux mixture, after administration the 18th day, only
There is Bordeaux mixture to also have certain protecting effect, other 7 kinds of medicaments all lose protecting effect completely.
The sprinkling of chemical agent will certainly bring hidden danger to the food-safety problem of fruits and vegetables, because apple is directly edible
Food, therefore can not avoid that chemical agent is remained on fruit body and chemical agent can penetrate into fruit body with the growth of plant
It is interior, it is food-safe to cause serious threat.
Another extremely serious leaf diseases of harm apple tree is alternaria leaf spot of apple, is one kind by alternaric bacteria apple
Fungal disease caused by fruit specialized form (Alternaria alternata f.sp.mali), the disease is the beginning of the fifties in Japan
It was found that, the subsequent seventies spreads to China, is also found in US and European to the nineties.The danger of alternaria leaf spot of apple
Evil rises year by year, is fallen ill in recent years in the apple producing region such as China Beijing, Shandong, Liaoning, Hebei extremely serious.The disease mainly infects
Apple tree new life blade causes early leaf fall, and the circular disease of a diameter of 2-3mm brown occurs in alternaria leaf spot of apple early stage
Spot, afterwards scab be gradually expanded into a diameter of 5-6mm bronzing scab, puce is presented in edge, and center usually has with one heart
Take turns line to occur, 3-5 scab occur on general blade will cause disease leaf to come off.The apple variety of the sick main harm has red
Star, marshal, blue or green perfume, Fuji etc., in recent years, the harm to apple varieties such as golden hat, state's light has risen.Alternaria leaf spot of apple
It is fungal disease, it can survive the winter on the fallen leaves of morbidity or tree, and conidium can be with air-flow after Second Year spring rain
Or wind and rain continues to propagate, and then infects blade from stomata.
Alternaria leaf spot of apple is prevented and treated at present, it usually needs with reference to Agro-chemicals control, conventional medicament mainly has
10% 1000-1500 times of Wettable polyoxin powder liquid, 25% Tebuconazole (sword power is led to) 3000-4000 times of liquid of wettable powder
Deng.The medicine such as Mancozeb and second manganese has certain preventive effect to disease.The research of biology preventing control method is mainly concentrated at present
On bacillus, such as Sun Yang demonstrates bacillus subtilis BS-3 15 by fruit tree leaf isolated experiment and apple spot is fallen
Leaf disease has good inhibiting effect, and Fu Xuechi etc. has arrived bacillus diseases prevention bacterial strain B-319 by field diseases prevention experiment screening
(Bacillus brevis) and B-9108 (Bacillus cereus), laboratory experiment combination field test draws B-319 and B-
9108 pairs of spot defoliation state of an illness can mitigate 60%-70%.
And for example the patent of Application No. 201410697783.3 discloses a kind of " Synergistic fertilizer for preventing and treating apple tree defoliation
Composition ", the invention discloses a kind of synergetic pesticide composition for preventing and treating apple tree defoliation, including quality proportioning is 100:1
~1:100 paclobutrazol and Tebuconazole.The synergetic pesticide composition for the preventing and treating apple tree defoliation that the invention is provided is to apple tree
Defoliation preventing and treating has certain synergistic effect, and delaying drug resistance development extends medicament service life.Application No.
201610570867.X patent discloses " prevention and controls of potted apple tree spot defoliation ", and the invention provides a kind of basin
The prevention and controls of apple peels and cores are planted, belong to pest and disease control of fruit trees field.The prevention and controls are comprised the step of：
(1) herbal raw material pre-treatment；(2) potted apple seedling leaching root processing；(3) prepared by compost；(4) transplant；(5) pour.This hair
Bright advantageous effects：The spot defoliation that the present invention greatly reduces potted apple tree occurs, while can also reduce basin
Apple peels and cores germ is planted to develop immunity to drugs degree.
Similarly, the sprinkling of chemical agent will certainly bring hidden danger to the food-safety problem of fruits and vegetables, food-safe to make
Into serious threat.In addition, periodically using for chemical agent, will certainly make the pathogen of crop develop immunity to drugs, so as to bring
Be chemical agent be excessively used or different chemical agent compounding use, and then food-safe problem brings bigger
Hidden danger.Meanwhile, chemical agent is irreversible to the pollution that the environment such as soil, water source are brought.
Biological control method is more advantageous compared with chemical prevention and control method, except biological agent production is easy, user
Just outer, the mix bacterium agent being made using biocontrol microorganisms and its metabolite is met more environment-friendly, pollution-free non agricultural chemical residuum
The strategic requirement of China's sustainable development.And biocontrol agent is small to environmental change susceptibility, effect stability, this is also its one
Big advantage.At present, though China reduces the use of chemical agent intentionally, in preventing and treating apple anthrax leaf blight and apple spot fallen leaves
Chemical agent is still the apple anthrax leaf blight that main means of prevention, especially China are newly broken out in disease, also without effective
Biological prevention and control agent, this cause for apple leaf diseases biocontrol agent exploitation it is particularly important.
This patent provide it is a kind of mainly for apple anthrax leaf blight and the biocontrol agent of alternaria leaf spot of apple, gram
The problems such as taking food security hidden danger that chemical agent brings and developed immunity to drugs to pathogen, pollution is produced to environment it is same
When, not only both diseases have significant prevention effect to the microbial inoculum to more than, and to the other diseases and fruits and vegetables of apple tree
The pathogenic bacteria of disease have significant fungistatic effect, can be applied to the preventing and treating of correspondingly diseases of garden stuff.
The content of the invention
The problem of present invention is solved is the defect for overcoming above-mentioned utilization Agro-chemicals control apple tree leaf diseases method, is carried
For one kind have the apple tree leaf portion fungal diseases such as preventing and treating apple anthrax leaf blight, alternaria leaf spot of apple and apple tree its
The biocontrol microorganisms of his disease --- cocoa streptomycete (Streptomyces cacaoi) FN, strepto- is prepared using cocoa streptomycete FN
The preventing and treating that bacterium biocontrol agent is applied to apple tree leaf diseases can not only substitute chemical prevention and control method completely, exclude food security hidden
Suffer from, and prominent effect is obtained in terms of the validity and antibacterial broad spectrum activity of preventing and treating apple tree leaf diseases.At the same time
The streptomycete possesses the characteristic of broad-spectrum antibacterial, can also prevent and treat other fruits and vegetables such as cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato
Etc. disease.
It is specific as follows by the preparation method of the cocoa streptomycete FN cocoa streptomycete FN biocontrol agents prepared：
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL。
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1-3% (v/v), 28 DEG C,
160-240r/min, Shaking culture 90-100h, obtains seed liquor.
Preferably, seed liquor culture：The spore suspension is inoculated into seed liquor culture by inoculum concentration 1-1.5% (v/v)
In base, 28 DEG C, 180-220r/min, Shaking culture 95-98h obtain seed liquor.
It is highly preferred that seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1% (v/v)
In, 28 DEG C, 200r/min, Shaking culture 96h obtain seed liquor.
Solid fermentation culture：The seed liquor is inoculated into water content by inoculum concentration volume mass than 5-15% (v/m)
In 50% solid fermentation culture medium, 28 DEG C of koji plate fermentation cultures 3-6 days.20-30 DEG C air-dries, and is ground with pulverizer, collects logical
The microbial inoculum of 120 mesh sieve is crossed, in the storage of room temperature shady place, cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN is obtained
The spawn activity of biocontrol agent is not less than 2 × 1011cfu/g。
Preferably, solid fermentation culture：The seed liquor is inoculated into by inoculum concentration volume mass than 8-12% (v/m) and contained
In the solid fermentation culture medium of water 50%, 28 DEG C of koji plate fermentation cultures 3-5 days start to terminating since fermentation, maintain solid to send out
The constant water content of ferment culture medium：50%, overturned 2-3 times during fermentation, treat fermentation ends, 25-30 DEG C air-dries, and is ground with pulverizer
Mill, collects the microbial inoculum by 120 mesh sieve, in the storage of room temperature shady place, obtains cocoa streptomycete FN biocontrol agents, the cocoa
The spawn activity of streptomycete FN biocontrol agents is not less than 2 × 1011cfu/g。
It is highly preferred that solid fermentation culture：The seed liquor is inoculated into by inoculum concentration volume mass than 10% (v/m) and contained
In the solid fermentation culture medium of water 50%, 28 DEG C of koji plate fermentation cultures 4 days start to terminating, maintenance solid fermentation since fermentation
The constant water content of culture medium：50%, overturned 3 times during fermentation, treat fermentation ends, 28 DEG C air-dry, and are ground with pulverizer, collect
By the microbial inoculum of 120 mesh sieve, in the storage of room temperature shady place, cocoa streptomycete FN biocontrol agents, the cocoa streptomycete are obtained
The spawn activity of FN biocontrol agents is not less than 2 × 1011cfu/g。
The 2216E solid mediums (g/L)：Bacto peptone No.25, dusty yeast 1, agar 20, surplus is distilled water,
PH is natural；
The seed liquid culture medium (g/L)：Corn flour 10, peptone 10, dusty yeast 5, dipotassium hydrogen phosphate 2, magnesium sulfate
0.3, surplus is distilled water, and pH is natural；
The solid fermentation culture medium (%) (m/m)：Rice husk 20%, wheat bran 44.7%, corn flour 22%, beancake powder
12%, calcium carbonate 0.8%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.2%, pH is natural.
The cocoa streptomycete FN biocontrol agents have prevention and control capability to fruits and vegetables fungal disease.
Further, the cocoa streptomycete FN biocontrol agents products application is in preventing and treating fruits and vegetables fungal disease.
Further, the fruits and vegetables fungal disease is apple tree fungal disease.
Further, the apple tree fungal disease is apple anthrax leaf blight, alternaria leaf spot of apple, apple decay
Disease, armillariella root rot of apple, ring rot of apple, apple phytophthora rot.
Further, the fruits and vegetables fungal disease is that cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, watermelon are withered
Wither disease, sclerotinia sclerotiorum, Cabbage Leaf Spot.
Cocoa streptomycete FN in the cocoa streptomycete FN biocontrol agents is specially cocoa streptomycete (Streptomyces
Cacaoi) FN, deposit number is CGMCC No.13080.
The cocoa streptomycete FN is isolated from the vegetable soil of Hebei province Funing County.Cocoa provided by the present invention
Streptomycete (Streptomyces cacaoi) FN is on October 12nd, 2016 in China Committee for Culture Collection of Microorganisms
Common micro-organisms center (CGMCC) preservation, numbering is CGMCC No.13080, and place is BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica.
The 16S rDNA sequences of the cocoa streptomycete FN are shown in sequence table SEQ ID No.1.
The cocoa streptomycete FN morphological features are：On 2216E solid mediums, mycelial growth is good, substrate mycelium
In yellow, aerial hyphae is in Slate grey；Synthesize slow-growing on an agar medium (ISP1) in Gao Shi, substrate mycelium is in shallow
Yellow, aerial hyphae is in light gray；The well-grown on brewer's wort yeast extract agar culture medium (ISP2), substrate mycelium is in Huang
Color, aerial hyphae is in Slate grey；At oatmeal agar medium (ISP3), substrate mycelium is in faint yellow, and aerial hyphae is white；
At inorganic salts starch agar medium (ISP4), substrate mycelium is in yellow, and aerial hyphae is in light gray；In glycerine asparagine fine jade
Fat culture medium (ISP5), substrate mycelium is in faint yellow, and aerial hyphae is white；Twist, spore is spherical in shape for fibrillae of spores；
The Gao Shi synthesizes agar medium (ISP1) composition：Soluble starch 20g/L, K2HPO40.5g/L,
KNO31g/L, MgSO4·7H2O 0.5g/L, FeSO4·7H2O 0.01g/L, agar 20g/L, surplus are distilled water, pH 7.2-
7.4；
The brewer's wort yeast extract agar culture medium (ISP2) constitutes：Dusty yeast 4g/L, malt flour 10g/L, glucose
4g/L, agar 20g/L, surplus are distilled water, pH 7.2-7.3；
The oatmeal agar medium (ISP3) constitutes：Oat 20g/L, Trace salts solution 1mL/L, agar 20g/L,
Surplus is distilled water, pH7.2；
The inorganic salts starch agar medium (ISP4) constitutes：Soluble starch 10g/L, K2HPO41g/L,
MgSO4·7H2O 1g/L, NaCl 1g/L, (NA4)2SO42g/L, CaCO32g/L, Trace salts solution 1mL/L, agar 20g/L,
Surplus is distilled water, pH 7.0-7.4；
The glycerine asparagine agar medium (ISP5) constitutes：Glycerine 10g/L, altheine 1g/L, it is micro
Salting liquid 1m/L, K2HPO41g/L, agar 20g/L, surplus are distilled water, and pH is natural；
Wherein described Trace salts solution, which is constituted, is：FeSO4·7H2O 0.1g, MnCl2·4H2O 0.1g, ZnSO4·7H2O
0.1g, distilled water 100mL, pH are natural.
Further, the physio-biochemical characteristics of the cocoa streptomycete FN are：Gelatin liquefaction, Starch Hydrolysis is strong, and milk is not
Solidification, does not produce hydrogen sulfide, does not produce melanin, do not reduce nitrate, using α-D- lactose, glycogen, D- cellobioses, sweet ammonia
Acyl-Pidolidone, methyl pyruvate, polysorbate40, Tween 80, PEARLITOL 25C, D, L- α-glycerine, N- acetylaminohydroxyphenylarsonic acid D- grapes amine,
D-Glucosaminic acid.
The cocoa streptomycete FN can suppress to cause the pathogenic bacteria of fruits and vegetables fungal disease.
Further, the pathogenic bacteria include anthrax leaf spoting bacteria, alternaric bacteria apple specialized form, Belém lattice grape seat chamber
The sharp spore of bacterium, apple black skin shell bacterium, Fusarium oxysporum, Phytophthora cactorum, the spore of watermelon shell two, Rhizoctonia solani Kuhn, sporulation, watermelon
The growth of various pathogens including sickle-like bacteria, sclerotinite, rape rod method etc..
The cocoa streptomycete FN has prevention and control capability to fruits and vegetables fungal disease.
Further, the fruits and vegetables fungal disease is apple tree fungal disease.
Further, the apple tree fungal disease includes apple tree leaf portion fungal disease.
Further, the apple tree leaf portion fungal disease includes apple anthrax leaf blight, alternaria leaf spot of apple.
Further, the apple tree fungal disease also include canker of apple fruit, armillariella root rot of apple, ring rot of apple,
Apple phytophthora rot.
Further, the fruits and vegetables fungal disease also includes cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, west
Cucurbit wilt, sclerotinia sclerotiorum, Cabbage Leaf Spot.
Applications of the cocoa streptomycete FN in preventing and treating fruits and vegetables fungal disease.
Further, applications of the cocoa streptomycete FN in preventing and treating apple tree fungal disease.
Further, the cocoa streptomycete FN is in preventing and treating apple anthrax leaf blight, alternaria leaf spot of apple, apple decay
Application in disease, armillariella root rot of apple, ring rot of apple, apple phytophthora rot.
Further, the cocoa streptomycete FN is in preventing and treating cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, watermelon
Application in droop, sclerotinia sclerotiorum, Cabbage Leaf Spot.
Beneficial effect：
1st, have aobvious to alternaria leaf spot of apple and apple anthrax leaf blight by the cocoa streptomycete FN biocontrol agents prepared
The prevention effect of work.
The field test of practical application shows, cocoa streptomycete FN biological controls microbial inoculum on preventing and treating apple spot fallen leaves,
When consumption is 1000,3000,5000 times of liquid of dilution, its prevention effect is respectively：87.10%, 85.58%, 82.74%.
In 1% and 5% level of difference, it is dilution 1000 that cocoa streptomycete FN biological controls microbial inoculum, which prevents and treats alternaria leaf spot of apple in consumption,
Prevention effect during times liquid, in consumption is 3000,5000 times of liquid better than it, and its prevention effect when diluting 1000 times of liquid is notable
In dosage it is dilution 1000 times of liquid and 80% Mancozeb wettable higher than the Wettable polyoxin powder of comparison medicament 10%
Prevention effect of the pulvis when dosage is 600 times of liquid of dilution.
Cocoa streptomycete FN biological controls microbial inoculum preventing and treating apple anthrax leaf it is withered on, when consumption for dilution 1000,3000,
During 5000 times of liquid, its prevention effect is respectively：82.17%, 77.83%, 73.24%.In 1% and 5% level of difference, cocoa
Streptomycete FN biological controls microbial inoculum prevents and treats prevention effect of the apple anthrax leaf blight when consumption is 1000 times of liquid of dilution, better than it
It is 3000,5000 times of liquid in consumption.At the same time, preventing and treating effect during 1000 times of liquid of cocoa streptomycete FN biological control agentses dilution agent
It is 1000 times of liquid of dilution and 80% Mancozeb that fruit, which is significantly higher than the Wettable polyoxin powder of comparison medicament 10% in dosage,
Prevention effect of the wettable powder when dosage is 600 times of liquid of dilution.
Do not find that cocoa streptomycete FN biocontrol agents have poisoning phenomenon during experiment.
2nd, the validity of preventing and treating apple tree leaf diseases, broad spectrum activity are strong：Cocoa streptomycete (Streptomyces cacaoi)
FN CGMCC NO.13080, can also be to other diseases of apple tree：Canker of apple fruit, armillariella root rot of apple, ring rot of apple, apple
Phytophthora rot also has very strong rejection ability.
3rd, validity, the broad spectrum activity for preventing and treating fungal disease on other fruits and vegetables are strong：Cocoa streptomycete FN is to other fruits and vegetables
Upper fungal disease：Cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, watermelon blight, sclerotinia sclerotiorum, Chinese cabbage blackspot
Disease has very strong rejection ability.
4th, cocoa streptomycete FN has broad-spectrum antibacterial to the pathogenic bacterium of fruits and vegetables, and experiment shows, cocoa streptomycete FN can be right
The pathogenic bacteria of fruits and vegetables are respectively provided with effective inhibitory action, are below bacteriostasis rates of the cocoa streptomycete FN to different pathogenic bacteria：Anthrax
Leaf spoting bacteria：86.2%th, alternaric bacteria apple specialized form；88.9%th, Botryosphaeria berengeriana：77.1%th, the black Beancurd sheet of apple
Shell bacterium：89.6%th, Fusarium oxysporum：80.9%th, Phytophthora cactorum：83.6%th, the spore of watermelon shell two：71.4%th, miliary damping-off：
74.0%th, sporulation：80.9%th, the sharp spore reaping hook of watermelon：77.9%th, sclerotinite：81.4%th, rape rod method：90.1%；
5th, higher genetic stability：Cocoa streptomycete FN is special to anthrax leaf spoting bacteria, rod method apple after passing for 30 generations
Change type, Botryosphaeria berengeriana, apple black skin shell bacterium, Fusarium oxysporum, Phytophthora cactorum, the spore of watermelon shell two, miliary damping-off, eggplant
The sharp spore reaping hook of alternaric bacteria, watermelon, sclerotinite, the bacteriostasis rate of rape rod method are respectively 85.3%, 88.0%, 76.5%,
88.5%th, 77.1%, 82.5%, 69.3%, 72.5%, 78.6%, 76.1%, 80.2%, 88.6%, with cocoa streptomycete FN
1st generation bacteriostasis rate is basically identical, shows that cocoa streptomycete FN of the present invention will not be reduced pair with the increase of passage number
The inhibitory activity of above-mentioned pathogen, with higher genetic stability.
6th, stronger heat resistance, acid and alkali resistance, higher ultraviolet-resistant capacity：Cocoa streptomycete FN has stronger resistance to
Hot, experiment shows, cocoa streptomycete FN can keep good growth conditions under conditions of 50 DEG C, and lethal temperature reaches 95
DEG C, thus illustrate, cocoa streptomycete FN still has higher bacterial strain vigor under high temperature stress.Cocoa streptomycete is to ultraviolet
Insensitive, after ultraviolet prolonged exposure 24h, bacterium survival rate is up to 77%, and after ultraviolet prolonged exposure 32h, Strain survival rate is still big
In 60%.
Cocoa streptomycete FN can not only keep stronger spawn activity during the fermentation, on the other hand alternatively
Bright, fermenting agent can not only keep significant fungistatic effect during the preventing and treating applied to pathogen under normal temperature condition,
And good bacterial strain vigor and inhibitory effect can be remained in that in hot weather.And under outdoor intense light irradiation weather,
Remain to keep good strain activity.
Cocoa streptomycete FN has acid resistance and extremely strong alkali resistance, under pH10 alkaline stress, is still presented good
Growth conditions, can not only keep stronger spawn activity during the fermentation, and pathogen is applied in fermenting agent
Preventing and treating during, extraneous source can also be resisted in the different acid or alkali environment such as agricultural chemicals, sleet, while keeping higher antibacterial
Vigor.
In addition, cocoa streptomycete FN has good salt resistance ability, the salt stress that salt ionic concentration is 17% can be resistant to
Effect.
7th, strain fermentation vigor is high：The invention provides cocoa streptomycete (Streptomyces cacaoi) FN CGMCC
The preparation of NO.13080 biocontrol agents, finally obtains bacterium vigor by liquid fermentation seed liquor and solid fermentation and is not less than 2.0
×10 11Cfu/g biocontrol agent, effect is very notable in the application of preventing and treating apple tree leaf diseases.
8th, controlling plant diseases are carried out using the cocoa streptomycete FN with antagonism and is not likely to produce the resistance to the action of a drug, wait excellent
Point, is the first choice of biological control.At the same time, cocoa streptomycete FN can avoid bringing hidden to the food-safety problem of fruits and vegetables
Suffer from, and serious threat is caused to health.Biocontrol agent production is convenient, easy to use, using cocoa streptomycete FN and
The mix bacterium agent that its fermentating metabolism product is made meets China's sustainable development to more environment-friendly, pollution-free non agricultural chemical residuum
The strategic requirement of exhibition.And biocontrol agent is small to environmental change susceptibility, effect stability.
It should be noted that the solution have the advantages that the knot that each processing step and parameter are mutually cooperateed with, interacted
Really, the superposition of not simple technique, the effect that the combination of each technique is produced is considerably beyond each single process function and effect
The superposition of fruit, with preferable advanced and practicality.
Brief description of the drawings
Fig. 1 is aerial hyphae and spore chain form (10 × 40 times) under cocoa streptomycete FN light microscopes；
After Fig. 2 is the normal mycelia of alternaria leaf spot of apple pathogen and spore shape and handled through cocoa streptomycete FN
Distortion mycelia and distortion spore shape, wherein 2a：Normal mycelia (10 × 10 times of mirrors), 2b：Normal spore (10 × 40 times of mirrors
Under), 2c：Distortion mycelia (10 × 10 times of mirrors), 2d：Distort spore (under 10 × 40 times of mirrors).
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
The various changes carried out on the premise of invention spirit and scope to the material component in these embodiments and consumption or change
Belong to protection scope of the present invention.
Cocoa streptomycete (Streptomyces cacaoi) FN's of embodiment 1 isolating and purifying and screening
Cocoa streptomycete 1. (Streptomyces cacaoi) FN isolation and purification
Soil sample is gathered from the vegetable garden of Funing to be fitted into sterile bag, takes 10g to add sterilized distilled water containing 100mL and glass
In the triangular flask of pearl, shaking table 200rpm concussion 30min are placed in, are stood after 10min, shaken up and contained with pipettor absorption 5mL additions
In the small triangular flask of 45mL sterilized waters, that is, dilute 102Times, gradient dilution is carried out successively to 10-3、10-4、10-5、10-6Times, respectively
100 μ l are drawn with pipettor to be coated on containing antibiotic (potassium bichromate 50mg/L, nalidixic acid 15mg/L and nystatin
On 2216E flat boards 50mg/L), each concentration sets 3 repetitions, is placed in 28 DEG C of incubators and cultivates 5-10 days.With sterilizing
Bamboo stick picking actinomyces single bacterium, which is fallen on the 2216E flat boards of Fresh, to be isolated and purified.
2. the screening of Antagonistic Fungi
Using apple anthrax leaf blight pathogen and alternaria leaf spot of apple pathogen as target bacterium, flat board opposite culture is utilized
Method carries out the screening of biocontrol bacterial strain, is crossed at the diameter 90mm flat boards back side, and right-angled intersection point is the flat board center of circle, on cross hairs
3 points therein away from 4 points at the 25mm of the center of circle are inoculated with 3 plants of 7mm probiotics bacteria cakes, the 7mm PDA trainings of remaining 1 point of inoculation blank respectively
Base is supported as control, by pathogen 7mm pure culture biscuits involvng inoculations in the PDA plate center of Fresh, 28 DEG C is placed in and cultivates 5-10 days, sieve
Choosing obtains, to apple anthrax leaf blight and the strong bacterial strain of alternaria leaf spot of apple bacteriostasis, being named as FN.
3. bacterial strain is identified
Morphological feature, physio-biochemical characteristics and the 16S rDNA the sequencing results of comprehensive FN bacterial strains, are accredited as
Cocoa streptomycete.Specific qualification result is as follows：
Cocoa streptomycete FN morphological features：On 2216E solid mediums, mycelial growth is good, substrate mycelium yellow, gas
Raw mycelia Slate grey；Gao Shi synthesis No. one (ISP1) is slow-growing, and substrate mycelium is light yellow, and aerial hyphae is light grey；Brewer's wort
Yeast extract agar (ISP2) well-grown, substrate mycelium yellow, aerial hyphae Slate grey；Oatmeal agar (ISP3) substrate mycelium
It is faint yellow, aerial hyphae white；Inorganic salts starch agar (ISP4) substrate mycelium yellow, aerial hyphae light gray；Glycerine Tianmen
Winter element agar (ISP5), substrate mycelium is faint yellow, aerial hyphae white；Fibrillae of spores spiral shape, spore is spherical as described in Figure 1.
Cocoa streptomycete FN physio-biochemical characteristics：Gelatin liquefaction, Starch Hydrolysis is strong, and milk does not solidify, and does not produce hydrogen sulfide, no
Melanin is produced, nitrate is not reduced, using α-D- lactose, glycogen, D- cellobioses, glycyl-L-glutamic acid, pyruvic acid first
Ester, polysorbate40, Tween 80, PEARLITOL 25C, D, L- α-glycerine, N- acetylaminohydroxyphenylarsonic acid D- grapes amine, D-Glucosaminic acid.
Cocoa streptomycete (Streptomyces cacaoi) FN is protected on October 12nd, 2016 in Chinese microorganism strain
Administration committee common micro-organisms center (CGMCC) preservation is hidden, numbering is CGMCC NO.13080.
The cocoa streptomycete FN of embodiment 2 growth characteristics
1. growth temperature
By 2216E fluid nutrient mediums (bacto peptone No.2 5g/L, dusty yeast 1g/L, surplus are distilled water, and pH is natural)
It is dispensed into cuvette, 121 DEG C of moist heat sterilization 30min, there must not be precipitation in nutrient solution.By cocoa streptomycete FN inoculations
Shaking flask 48 is inoculated into the test tube of the fluid nutrient medium containing 2216E respectively as Inoculant by 1 ‰ in 2216E fluid nutrient mediums, right
According to not being inoculated with.Respectively in 0 DEG C, 7 DEG C, 15 DEG C, 20 DEG C, 28 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C of incubator
Culture 3d-5d is carried out, if 3 repetitions, observes and records growth (precipitation) situation.
2. resistance to acids and bases
By 2216E fluid nutrient mediums be adjusted to dilute HCl or NaOH different pH (1.0,1.5,2.0,2.5,3.0,3.5,
, 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0,11.5,12.0), point 4.0th, 4.5
It is attached in cuvette, 121 DEG C of moist heat sterilization 30min.2216E liquid by 1 ‰ inoculation cocoa streptomycetes to FN to different pH
In culture medium, control is not inoculated with, if 3 repetitions.28 DEG C are cultivated 3-5 days, record strain growth (precipitation) situation.
3. salt tolerance
Add different amounts of NaCl in 2216E fluid nutrient mediums again, be configured to salinity for 1%, 2%, 5%, 7%,
9%th, 11%, 13%, 15%, 17%, 20%, 25%, 30% 2216E fluid nutrient mediums, are dispensed into cuvette, 121
DEG C moist heat sterilization 30min.By 1 ‰ inoculation cocoa streptomycete FN into the 2216E fluid nutrient mediums of different salinity, control does not connect
Kind, if 3 repetitions.28 DEG C are cultivated 3-5 days, record strain growth (precipitation) situation.
4. lethal temperature
By cocoa streptomycete FN bacterium solutions be respectively put into 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C, 95 DEG C, 100
DEG C water-bath in, be immediately placed in cold water and cool down rapidly after timing 10min, if 3 repetitions.Take 100 μ l coatings fresh respectively
2216E flat boards on, be placed in 28 DEG C cultivate 3-5 days, record strain growth situation.
5. sensitivity to ultraviolet light
Take the μ l of cocoa streptomycete FN zymotic fluids 100 to be applied on 2216E flat boards, be placed at 30w uviol lamps 40cm respectively according to
Penetrate after 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 16h, 20h, 24h, 32h, 40h, 48h, be placed in 28 DEG C and cultivate 3-5 days, record bacterium
Strain growing state.
Cocoa streptomycete growth characteristics are shown in Table 1, and optimum growth temperature is 28 DEG C, and growth temperature range is 15 DEG C -45 DEG C；Most
Suitable growth pH7.0, growth pH scopes are 3.0-12.0；Resistance to 15% salt ionic concentration.Lethal temperature is 85 DEG C, 10min.To purple
Outside line is insensitive, after ultraviolet prolonged exposure 24h, and bacterium survival rate is up to 77%, and after ultraviolet prolonged exposure 32h, bacterium survival rate is still
More than 60%.
The cocoa streptomycete FN growth characteristics of table 1
Note：+ positive；++ strong positive；W* weakly positives；- negative
As can be seen from Table 1, cocoa streptomycete FN has stronger heat resistance, can keep good under conditions of 50 DEG C
Growth conditions, lethal temperature reaches 95 DEG C, thus illustrates, cocoa streptomycete FN still has higher bacterium under high temperature stress
Strain vigor.Insensitive to ultraviolet, after ultraviolet prolonged exposure 24h, bacterium survival rate is up to 77%, after ultraviolet prolonged exposure 32h,
Bacterium survival rate is still greater than 60%.
Cocoa streptomycete FN can not only keep stronger spawn activity during the fermentation, on the other hand alternatively
Bright, fermenting agent can not only keep significant fungistatic effect during the preventing and treating applied to pathogen under normal temperature condition,
And can high temperature weather, remain in that good bacterial strain vigor and inhibitory effect.And in outdoor intense light irradiation weather
Under, remain to keep good strain activity.
Cocoa streptomycete FN has acid resistance and extremely strong alkali resistance, under pH10 alkaline stress, is still presented good
Growth conditions, can not only keep stronger spawn activity during the fermentation, and pathogen is applied in fermenting agent
Preventing and treating during, extraneous source can also be resisted in the different acid or alkali environment such as agricultural chemicals, sleet, while keeping higher antibacterial
Vigor.
In addition, cocoa streptomycete FN has good salt resistance ability, the salt stress that salt ionic concentration is 17% can be resistant to
Effect.
Applications of the cocoa streptomycete FN of embodiment 3 to preventing and treating fruits and vegetables fungal disease
Cocoa streptomycete FN is determined to apple anthrax leaf blight, alternaria leaf spot of apple, ring rot of apple, apple decay
Disease, armillariella root rot of apple, apple phytophthora rot, cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, watermelon blight, rape sclerotium
Disease, the bacteriostasis rate of Cabbage Leaf Spot pathogen.Crossed at the diameter 90mm PDA plate back side plate center draw straight line, on line away from
2 points inoculation 7mm probiotics bacteria cakes at the 25mm of center, compare plating 7mm blank PDA culture mediums.By pathogen 7mm bacterium
Cake inoculation PDA plate center, is placed in 28 DEG C and cultivates 5-10 days, control pathogen radius and processing pathogen radius are counted respectively,
Calculate bacteriostasis rate.Bacteriostasis rate %=(control pathogen radius mm- processing pathogens radius mm)/(control pathogen radius mm- diseases
Opportunistic pathogen bacteria cake radius 3.5mm) × 100%.Cocoa streptomycete FN is subjected to Secondary Culture, and determines the 10th generation, the 15th generation, the 25th
Generation, in the 30th generation, experimental method was ibid to the bacteriostasis rate of fruits and vegetables fungoid disease opportunistic pathogen.
Bacteriostasis rate of the cocoa streptomycete FN of the table 2 difference algebraically to 12 kinds of plant disease pathogens
As can be seen from Table 2, cocoa streptomycete FN to Disease of Apple Trees (apple anthrax leaf blight, alternaria leaf spot of apple,
Canker of apple fruit, armillariella root rot of apple, ring rot of apple, apple phytophthora rot) there is very strong rejection ability, in addition, to other
Fungal disease (cucumber blight dis-ease, cucumber rhizoctonia rot, early blight of tomato, watermelon blight, sclerotinia sclerotiorum, Chinese cabbage on fruits and vegetables
Black spot) rejection ability it is also very strong.
Cocoa streptomycete FN is after passing for 30 generations, to anthrax leaf spoting bacteria, rod method apple specialized form, Belém lattice grape seat chamber
The sharp spore sickle of bacterium, apple black skin shell bacterium, Fusarium oxysporum, Phytophthora cactorum, the spore of watermelon shell two, miliary damping-off, sporulation, watermelon
Knife, sclerotinite, the bacteriostasis rate of rape rod method are respectively 85.3%, 88.0%, 76.5%, 88.5%, 77.1%, 82.5%,
69.3%th, 72.5%, 78.6%, 76.1%, 80.2%, 88.6%, it is basically identical with cocoa streptomycete FN 1st generation bacteriostasis rates,
Show that cocoa streptomycete FN of the present invention will not reduce the inhibitory activity to above-mentioned pathogen with the increase of passage number,
With higher genetic stability.
The antibacterial broad spectrum activities of the cocoa streptomycete FN of embodiment 4 determine the influence to pathogen mycelia and spore
With alternaria leaf spot of apple pathogen rod method apple transformant (Alternaria alternaria f.sp
Mali exemplified by).Alternaria leaf spot of apple pathogen is inoculated into PDA plate center, connect respectively according to both sides at the 25mm of flat board center
Probiotics is planted, 28 DEG C grow 7 days.Cut from the flat board grown rectangular close to probiotics and with apple spot fallen leaves
The fungus block of sick pathogen, by thin layer under the fungus block small blade cut, is placed on slide, is carried out by light microscope
Observation.The alternaria leaf spot of apple pathogen for not being inoculated with probiotics carries out same treatment.
By observation by light microscope alternaria leaf spot of apple pathogen mycelia and spore shape topology discovery, close to prebiotic
The mycelia of the pathogen at bacterium edge is distorted, it is impossible to form normal spore (Fig. 2).It is probably that cocoa streptomycete FN is produced
Metabolite be diffused by PDA plate, pathogen contact metabolite after lure that hypha form changes into, mycelia is swollen
Greatly, it is impossible to form normal spore.
The preparation of the cocoa streptomycete FN biocontrol agents of embodiment 5
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1% (v/v), 28 DEG C,
200r/min, Shaking culture 96h, obtains seed liquor；
Solid fermentation culture：The seed liquor is inoculated into water content 50% by inoculum concentration volume mass than 10% (v/m)
Solid fermentation culture medium in, 28 DEG C of koji plate fermentation cultures 4 days start to terminating since fermentation, maintenance solid fermentation culture medium
Constant water content：50%, overturned 3 times during fermentation, treat fermentation ends, 28 DEG C air-dry, and are ground with pulverizer, collect and pass through 120
The microbial inoculum of mesh sieve, in the storage of room temperature shady place, obtains cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN biocontrol microorganisms
The spawn activity 3.7 × 10 of agent11cfu/g。
The 2216E solid mediums (g/L)：Bacto peptone No.25, dusty yeast 1, agar 20, surplus is distilled water,
PH is natural；
The seed liquid culture medium (g/L)：Corn flour 10, peptone 10, dusty yeast 5, dipotassium hydrogen phosphate 2, magnesium sulfate
0.3, surplus is distilled water, and pH is natural；
The solid fermentation culture medium (%) (m/m)：Rice husk 20%, wheat bran 44.7%, corn flour 22%, beancake powder
12%, calcium carbonate 0.8%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.2%, pH is natural.
The culture medium of example 6 below -9 is identical with embodiment 4.
The preparation of the cocoa streptomycete FN biocontrol agents of embodiment 6
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1% (v/v), 28 DEG C,
160r/min, Shaking culture 90h, obtains seed liquor；
Solid fermentation culture：The seed liquor is inoculated into water content than 15% (v/m) by inoculum concentration volume mass is
In 50% (v/m) solid fermentation culture medium, 28 DEG C of koji plate fermentation cultures 3 days.20 DEG C air-dry, and are ground with pulverizer, collect logical
The microbial inoculum of 120 mesh sieve is crossed, in the storage of room temperature shady place, cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN is obtained
The spawn activity 2.2 × 10 of biocontrol agent11cfu/g。
The preparation of the cocoa streptomycete FN biocontrol agents of embodiment 7
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 3% (v/v), 28 DEG C,
240r/min, Shaking culture 100h, obtains seed liquor；
Solid fermentation culture：The seed liquor is inoculated into water content 50% by inoculum concentration volume mass than 5% (v/m)
In solid fermentation culture medium, 28 DEG C of koji plate fermentation cultures 6 days.30 DEG C air-dry, and are ground with pulverizer, collect and pass through 120 mesh sieve
Microbial inoculum, in room temperature shady place storage, obtain cocoa streptomycete FN biocontrol agents, the bacterium of the cocoa streptomycete FN biocontrol agents
Plant vigor 2.3 × 1011cfu/g。
The preparation of the cocoa streptomycete FN biocontrol agents of embodiment 8
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1% (v/v), 28 DEG C,
180r/min, Shaking culture 95h, obtains seed liquor；
Solid fermentation culture：The seed liquor is inoculated into water content 50% by inoculum concentration volume mass than 8% (v/m)
In solid fermentation culture medium, 28 DEG C of koji plate fermentation cultures 3 days start to terminating, the perseverance of maintenance solid fermentation culture medium since fermentation
Determine water content：50%, overturned 2 times during fermentation, treat fermentation ends, 25 DEG C air-dry, and are ground with pulverizer, collect and pass through 120 mesh
The microbial inoculum of sieve, in the storage of room temperature shady place, obtains cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN biocontrol agents
Spawn activity 3.2 × 1011cfu/g。
The preparation of the cocoa streptomycete FN biocontrol agents of embodiment 9
It is prepared by spore suspension：On the inclined-plane that sterilized water is added to the 2216E solid mediums for preserving cocoa streptomycete FN,
And then spore is scraped off, concussion is mixed, and obtains spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1.5% (v/v), 28 DEG C,
220r/min, Shaking culture 98h, obtains seed liquor；
Solid fermentation culture：The seed liquor is inoculated into water content 50% by inoculum concentration volume mass than 12% (v/m)
Solid fermentation culture medium in, 28 DEG C of koji plate fermentation cultures 5 days start to terminating since fermentation, maintenance solid fermentation culture medium
Constant water content：50%, overturned 3 times during fermentation, treat fermentation ends, 30 DEG C air-dry, and are ground with pulverizer, collect and pass through 120
The microbial inoculum of mesh sieve, in the storage of room temperature shady place, obtains cocoa streptomycete FN biocontrol agents, the cocoa streptomycete FN biocontrol microorganisms
The spawn activity 3.5 × 10 of agent11cfu/g。
The cocoa streptomycete FN biological controls microbial inoculum of embodiment 10 prevents and treats the field test of alternaria leaf spot of apple
Cocoa streptomycete FN biocontrol agents are prepared by method described in embodiment 5.
Field experimental design：Totally 6 processing, you can 1000 times of dilutions of streptomycete FN biocontrol agents；Cocoa streptomycete
3000 times of dilutions of FN biocontrol agents；Cocoa streptomycete FN 5000 times of dilutions of biocontrol agent；10% polyoxin wettable powder
1000 times of dilutions of agent；80% 600 times of mancozeb wettable powder dilution；Above-mentioned diluent liquid is clear water, control group
For clear water.Each processing is repeated 4 times, and adds up to 24 cells, per 2 apple trees of cell, random district's groups arrangement.Test site is in Shan
Xi Sheng Baojis Qianyang County Cao Bi towns Qu Jiawan villages apple orchard is carried out, and examination tree kind is " Fuji ", the age of tree 12 years, fruit tree spacing in the rows
3m, line-spacing 4m.Cocoa streptomycete FN biocontrol agents are that described in embodiment 6 prepared by method, are diluted and are sprayed after debita spissitudo with clear water
Apply.
Dispenser amounts to 6 times, May 18 first time, second of May 30, third time June 12, the 4th July 4.The
Five July 17, the 6th August 3 days.It is uniform using complete stool cauline leaf using the luxuriant DFH-16A types knapsack manual sprayer in Beijing
Spray-on process is handled.Spouting liquid is uniformly to squirt leave dual sides, and it is degree to have drop under decoction.
Investigation method：Investigation after 10-14 days before May 16 medicine and after August last medicine on the 3rd.It is equal per 2 plants of trees of cell
Investigation, every plant of point East, West, South, North, in 5 directions fix 2 young sprouts, investigate its whole blade and calculate disease index with
Prevention effect.Leaf portion disease level grade scale is as follows：
0 grade：Disease-free spot；
1 grade：Lesion area accounts for less than the 10% of whole leaf area；
3 grades：Lesion area accounts for the 11%-25% of whole leaf area；
5 grades：Lesion area accounts for the 26%-40% of whole leaf area；
7 grades：Lesion area accounts for the 41%-65% of whole leaf area；
9 grades：Lesion area accounts for more than the 66% of whole leaf area.
Drug effect computational methods：Disease index=100 × ∑ [(the sick numbers of sheets at different levels × relative value of series)]/(total blade of investigation
Number × 9)；Prevention effect (%)=[1- (disease index after disease index × treatment region medicine before the medicine of check plot)/(after the medicine of check plot
Disease index before disease index × treatment region medicine)] × 100%.Utilize the poor method of inspection logarithm of the new multipoles of the Duncan ' s of DPS softwares
According to being analyzed, as a result as shown in table 3.
Prevention effect of the cocoa streptomycete FN biocontrol agents of table 3 to alternaria leaf spot of apple
Note:The significance of difference of the lowercase letter in 5% level, capitalization represents the difference in 1% level
Conspicuousness.
The cocoa streptomycete FN biocontrol agents prepared by method described in embodiment 4, are diluted after 1000 times, uniformly with clear water
Spray to drip in the case where fruit tree up to blade has decoction and be defined, start to spray in the spring tip, spray altogether 6 times, to alternaria leaf spot of apple
Preventive effect is up to 87.10%.
It should be noted that：Cocoa streptomycete FN biological controls microbial inoculum prepared by 6-9 of the embodiment of the present invention equally has upper
State between experiment effect, each embodiment and little with above-mentioned experiment effect otherness.
The cocoa streptomycete biocontrol agent of embodiment 11 prevents and treats the field test of apple anthrax leaf blight
Cocoa streptomycete FN biocontrol agents are prepared by method described in embodiment 5.
Field experimental design：Totally 6 processing, cocoa streptomycete FN 1000 times of dilutions of biocontrol agent；Cocoa streptomycete FN
3000 times of dilutions of biocontrol agent；Cocoa streptomycete FN 5000 times of dilutions of biocontrol agent；10% Wettable polyoxin powder
1000 times of dilutions；80% 600 times of mancozeb wettable powder dilution；Above-mentioned diluent liquid is clear water, and control group is
Clear water.Each processing is repeated 4 times, and adds up to 24 cells, per 2 apple trees of cell, random district's groups arrangement.Test site is in Shaanxi
Province Baoji Qianyang County Cao Bi towns Qu Jiawan villages apple orchard is carried out, and examination tree kind is " gold hat ", the age of tree 15 years, fruit tree spacing in the rows 3m,
Line-spacing 4m.Cocoa streptomycete FN biocontrol agents are that described in embodiment 6 prepared by method, are sprayed after diluting debita spissitudo with clear water.
Dispenser amounts to 6 times, May 16 first time, second of June 1, third time June 13, the 4th July 5.The
Five July 17, the 6th August 7 days.It is uniform using complete stool cauline leaf using the luxuriant DFH-16A types knapsack manual sprayer in Beijing
Spray-on process is handled.Spouting liquid is uniformly to squirt leave dual sides, and it is degree to have drop under decoction.
Investigation method：Before medicine anthrax leaf blight does not occur for May 16.In investigation after 10-14 days after August last medicine on the 7th.
Investigate per 2 plants of trees of cell, every plant of point East, West, South, North, in 5 directions fix 2 young sprouts, investigate its whole blade and count
Calculate disease index and prevention effect.Leaf portion disease level grade scale is as follows：
0 grade：Disease-free spot；
1 grade：Lesion area accounts for less than the 10% of whole leaf area；
3 grades：Lesion area accounts for the 11%-25% of whole leaf area；
5 grades：Lesion area accounts for the 26%-40% of whole leaf area；
7 grades：Lesion area accounts for the 41%-65% of whole leaf area；
9 grades：Lesion area accounts for more than the 66% of whole leaf area.
Drug effect computational methods：Disease index=100 × ∑ [(the sick numbers of sheets at different levels × relative value of series)]/(total blade of investigation
Number × 9)；Prevention effect (%)=[1- (disease index after disease index × treatment region medicine before the medicine of check plot)/(after the medicine of check plot
Disease index before disease index × treatment region medicine)] × 100%.Utilize the poor method of inspection logarithm of the new multipoles of the Duncan ' s of DPS softwares
According to being analyzed, as a result as shown in table 4.
Prevention effect of the cocoa streptomycete FN biocontrol agents of table 4 to apple anthrax leaf blight
The cocoa streptomycete FN biocontrol agents prepared by method described in embodiment 4, are diluted after 1000 times, uniformly with clear water
It is degree to spray the drop in the case where fruit tree up to blade has decoction, starts to spray in the spring tip, sprays altogether 6 times, to apple anthrax leaf blight
Preventive effect is up to 82.17%.
Table of field test results by embodiment 9, embodiment 10 is bright, cocoa streptomycete FN biological controls microbial inoculum preventing and treating apple
Set spot fallen leaves and anthrax leaf is withered when consumption is 1000,3000,5000 times of liquid of dilution, to apple tree safety without poisoning.
Cocoa streptomycete FN biological controls microbial inoculum preventing and treating apple spot fallen leaves on, when consumption for dilution 1000,3000,
During 5000 times of liquid, its prevention effect is respectively：87.10%, 85.58%, 82.74%.In 1% and 5% level of difference, cocoa
Streptomycete FN biological controls microbial inoculum prevents and treats prevention effect of the alternaria leaf spot of apple when consumption is 1000 times of liquid of dilution, better than it
It is 3000,5000 times of liquid in consumption, and prevention effect during its 1000 times of liquid of dilution is significantly higher than comparison medicament anti-mildew more than 10%
Plain wettable powder is that 1000 times of liquid of dilution and 80% mancozeb wettable powder are 600 times of dilution in dosage in dosage
Prevention effect during liquid.
Cocoa streptomycete FN biological controls microbial inoculum preventing and treating apple anthrax leaf it is withered on, when consumption for dilution 1000,3000,
During 5000 times of liquid, its prevention effect is respectively：82.17%, 77.83%, 73.24%.In 1% and 5% level of difference, cocoa
Streptomycete FN biological controls microbial inoculum prevents and treats prevention effect of the apple anthrax leaf blight when consumption is 1000 times of liquid of dilution, better than it
It is 3000,5000 times of liquid in consumption.At the same time, preventing and treating effect during 1000 times of liquid of cocoa streptomycete FN biological control agentses dilution agent
It is 1000 times of liquid of dilution and 80% Mancozeb that fruit, which is significantly higher than the Wettable polyoxin powder of comparison medicament 10% in dosage,
Prevention effect of the wettable powder when dosage is 600 times of liquid of dilution.
1000 times of dilutions of cocoa streptomycete FN biological control microbial inoculums are withered to alternaria leaf spot of apple and apple anthrax leaf
Disease has good prevention effect, and better than 10% Polyoxin wettable powder of comparison medicament, 1000 times of liquid and 80% maneb
600 times of liquid of zinc wettable powder.Do not find that cocoa streptomycete FN biocontrol agents have poisoning phenomenon during experiment.
It should be noted that：Cocoa streptomycete FN biological controls microbial inoculum prepared by 6-9 of the embodiment of the present invention equally has upper
State between experiment effect, each embodiment and little with above-mentioned experiment effect otherness.
The heat endurance of the cocoa streptomycete FN biological control microbial inoculums of embodiment 12
Cocoa streptomycete FN biological controls microbial inoculum prepared by embodiment 5 is at a temperature of 50,70,90,100,121 DEG C
30min is handled, it is to be measured with 100 times of sterilized water dilution respectively.Using alternaria leaf spot of apple pathogen as subjects, by 7mm diseases
Opportunistic pathogen pure culture biscuits involvng inoculation is crossed plate center line, beaten on line away from 2 points at the 25mm of center with card punch to PDA plate center
7mm holes, are separately added into 100 μ l prepare liquids, and control adds 100 μ l sterilized waters.It is placed in 28 DEG C to cultivate 7-10 days, statistics control respectively
Pathogen radius and processing pathogen radius, calculate bacteriostasis rate.Bacteriostasis rate %=(control pathogen radius mm- processing pathogens
Radius mm)/(control pathogen radius mm- pathogen bacteria cakes radius 3.5mm) × 100%.
The thermal stability determination of the cocoa streptomycete FN biological control microbial inoculums of table 5
As can be seen from Table 5, cocoa streptomycete FN biological control microbial inoculums are after high-temperature process, and bacteriostasis does not change significantly
Become.From bacteriostasis rate as can be seen that microbial inoculum is after different high-temperature process, it is declined slightly after only 100 DEG C and 121 DEG C processing 30min,
But remain to reach more than 83.7%.It can be seen that, the antibacterial activity of cocoa streptomycete FN biological control microbial inoculums has stronger heat steady
It is qualitative.
It should be noted that：Cocoa streptomycete FN biological controls microbial inoculum prepared by 6-9 of the embodiment of the present invention equally has upper
State between experiment effect, each embodiment and little with above-mentioned experiment effect otherness.
SEQUENCE LISTING
<110>Shaanxi Feng Dan belles bio tech ltd
<120>A kind of preparation method for the biocontrol agent for preventing and treating apple tree fungal disease
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1490
<212> DNA
<213>Cocoa streptomycete FN（Streptomyces cacaoi）CGMCC No.13080
<400> 1
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60
gatgaaccgg tttcggccgg ggattagtgg cgaacgggtg agtaacacgt gggcaatctg 120
ccctgcactc tgggacaagc cctggaaacg gggtctaata ccggatatga ccaccggccg 180
catggtctgg tggtggaaag ctccggcggt gcaggatgag cccgcggcct atcagcttgt 240
tggtggggtg atggcctacc aaggcgacga cgggtagccg gcctgagagg gcgaccggcc 300
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattgcac 360
aatgggcgca agcctgatgc agcgacgccg cgtgagggat gacggccttc gggttgtaaa 420
cctctttcag cagggaagaa gcgcgagtga cggtacctgc agaagaagca ccggctaact 480
acgtgccagc agccgcggta atacgtaggg tgcgagcgtt gtccggaatt attgggcgta 540
aagagctcgt aggcggcctg tcgcgtcgga tgtgaaagcc cggggcttaa ccccgggtct 600
gcgttcgata cgggcaggct agagttcggc aggggagatt ggaattcctg gtgtagcggt 660
gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg gccgatactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
taaacgttgg gcactaggtg tgggcggcat tccacgtcgt ccgtgccgca gctaacgcat 840
taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggc 900
ccgcacaagc ggcggagcat gtggcttaat tcgacgcaac gcgaagaacc ttaccaaggc 960
ttgacataca tcggaaaact ctggagacag ggtccccctt tgggtcggtg tacaggtggt 1020
gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttatcctg tgttgccagc atgcctttcg gggtgatggg gactcacggg agactgccgg 1140
ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat gtcttgggct 1200
gcacacgtgc tacaatggcc ggtacaatga gctgcgatgc cgtgaggtgg agcgaatctc 1260
aaaaagccgg tctcagttcg gattggggtc tgcaactcga ccccatgaag tcggagtcgc 1320
tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt acacaccgcc 1380
cgtcacgtca cgaaagtcgg taacacccga agccggtggc ccaacccctt gtgggaggga 1440
attgtcgaag gtgggactgg cgattgggac gaagtcgtaa caaggtaacc 1490
Claims (6)
1. a kind of preparation method for the biocontrol agent for preventing and treating apple tree fungal disease, it is characterised in that specific as follows：
It is prepared by spore suspension：Sterilized water is added and preserved on the inclined-plane of 2216E solid mediums of cocoa streptomycete, and then is scraped
Spore is removed, concussion is mixed, obtain spore suspension, the cell concentration of the spore suspension is 108-109cfu/mL；
Seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1-3%, 28 DEG C, 160-240r/
Min, Shaking culture 90-100h, obtains seed liquor；
Solid fermentation culture：The seed liquor is sent out by inoculum concentration volume mass than the 5-15% solids for being inoculated into water content 50%
In ferment culture medium, 28 DEG C of koji plate fermentation cultures 3-6 days, 20-30 DEG C air-dries, and is ground with pulverizer, collects by 120 mesh sieve
Microbial inoculum, in the storage of room temperature shady place, obtains cocoa streptomycete biocontrol agent, the spawn activity of the cocoa streptomycete biocontrol agent
Not less than 1 × 1011cfu/g；
The biocontrol microorganisms are specially cocoa streptomycete (Streptomyces cacaoi) FN, and deposit number is CGMCC
No.13080。
2. a kind of preparation method of biocontrol agent for preventing and treating apple tree fungal disease as claimed in claim 1, its feature exists
In：
The seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1-1.5%, 28 DEG C,
180-220r/min, Shaking culture 95-98h, obtains seed liquor.
3. a kind of preparation method of biocontrol agent for preventing and treating apple tree fungal disease as claimed in claim 2, its feature exists
In：
The seed liquor culture：The spore suspension is inoculated into seed liquid culture medium by inoculum concentration 1%, 28 DEG C, 200r/
Min, Shaking culture 96h, obtains seed liquor.
4. a kind of preparation method of biocontrol agent for preventing and treating apple tree fungal disease as claimed in claim 1, its feature exists
In：
The solid fermentation culture：The seed liquor is inoculated into consolidating for water content 50% by inoculum concentration volume mass than 8-12%
In body fermentation medium, 28 DEG C of koji plate fermentation cultures 3-5 days start to terminating, the perseverance of maintenance solid fermentation culture medium since fermentation
Determine water content：50%, overturned 2-3 times during fermentation, treat fermentation ends, 25-30 DEG C air-dries, and is ground with pulverizer, collection passes through
The microbial inoculum of 120 mesh sieve, in the storage of room temperature shady place, obtains cocoa streptomycete FN biocontrol agents.
5. a kind of preparation method of biocontrol agent for preventing and treating apple tree fungal disease as claimed in claim 4, its feature exists
In：
The solid fermentation culture：The solid that the seed liquor is inoculated into water content 50% by inoculum concentration volume mass than 10%
In fermentation medium, 28 DEG C of koji plate fermentation cultures 4 days start to terminating, maintenance the constant of solid fermentation culture medium contains since fermentation
Water：50%, overturned 3 times during fermentation, treat fermentation ends, 28 DEG C air-dry, and are ground with pulverizer, collect and pass through 120 mesh sieve
Microbial inoculum, in room temperature shady place storage, obtain cocoa streptomycete FN biocontrol agents.
6. a kind of preparation method of biocontrol agent for preventing and treating apple tree fungal disease as claimed in claim 1, its feature exists
In：
2216E solid mediums composition in terms of g/L is：Bacto peptone No.2 5, dusty yeast 1, agar 20, surplus are steaming
Distilled water, pH is natural；
Seed liquid culture medium composition in terms of g/L is：Corn flour 10, peptone 10, dusty yeast 5, dipotassium hydrogen phosphate 2, sulfuric acid
Magnesium 0.3, surplus is distilled water, and pH is natural；
The solid fermentation culture medium mass percent is constituted：Rice husk 20%, wheat bran 44.7%, corn flour 22%, beancake powder
12%, calcium carbonate 0.8%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.2%, pH is natural.
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| CN109913384A (en) * | 2018-12-20 | 2019-06-21 | 蚌埠学院 | The preparation method and application of a kind of white light gray streptomycete and its biocontrol agent |
| CN112586260A (en) * | 2020-12-16 | 2021-04-02 | 张开银 | Apple orchard ecological planting method |
| CN113632679A (en) * | 2021-09-03 | 2021-11-12 | 重庆文理学院 | Disease-preventing cultivation method for kiwi fruits |
| CN116267923A (en) * | 2023-02-03 | 2023-06-23 | 陕西枫丹百丽生物科技有限公司 | Biological medicine patch for preventing and treating apple tree diseases and preparation method and application thereof |
| CN112961784B (en) * | 2021-02-18 | 2023-06-23 | 西北农林科技大学 | Endophytic Alternaria alternata Aa-Lcht and application thereof in preventing and treating apple tree rot |
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| CN113632679A (en) * | 2021-09-03 | 2021-11-12 | 重庆文理学院 | Disease-preventing cultivation method for kiwi fruits |
| CN116267923A (en) * | 2023-02-03 | 2023-06-23 | 陕西枫丹百丽生物科技有限公司 | Biological medicine patch for preventing and treating apple tree diseases and preparation method and application thereof |
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| CN106967635B (en) | 2020-09-25 |
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