CN106906172A

CN106906172A - One plant of Streptomycesalbidoflhaving and its application in terms of apple tree canker preventing and treating

Info

Publication number: CN106906172A
Application number: CN201710308301.4A
Authority: CN
Inventors: 李玲娣; 王灵敏; 孙振娜; 严立恩; 李学辉; 周天慧; 王江; 王楠
Original assignee: Shaanxi Fengdan Baili Biotechnology Co ltd
Current assignee: Shaanxi Fengdan Baili Biotechnology Co ltd
Priority date: 2017-05-04
Filing date: 2017-05-04
Publication date: 2017-06-30
Anticipated expiration: 2037-05-04
Also published as: CN106906172B

Abstract

The invention discloses a kind of Streptomycesalbidoflhaving Actin 1, can be grown by nutrition of Pathogen of Apple Canker mycelium, breeding, colonized for a long time at apple tree canker spot beneficial to biocontrol microorganisms, play long-term biological control effect, induce simultaneously and itself produce various extracellular cytohydrolists, pathogen cell disruption is made by the molten effect of coordinated enzyme, and stronger antibacterial substance can be produced, bacteriostasis rate to Pathogen of Apple Canker is 89.82%, there is preferable fungistatic effect to various pathogens such as Botryosphaeria berengeriana, bacteriostasis rate is 76.08 87.10%, with broad-spectrum antibacterial；It is main biocontrol microorganisms with the bacterium; carry out preventing and treating the fruits and vegetables pathogen such as apple tree canker; with prevention effect good (100%), efficiency high, recurrence rate low (0), accommodative ability of environment is strong, stabilization is strong, be not likely to produce the multiple advantage such as the resistance to the action of a drug, to improve the fruits and vegetables pathogens such as apple tree canker prevention effect, prevent pathogen from recurring, environmental protection it is significant.

Description

One plant of Streptomycesalbidoflhaving and its application in terms of apple tree canker preventing and treating

Technical field

The present invention relates to biocontrol microorganisms, and in particular to one plant of Streptomycesalbidoflhaving and its in terms of apple tree canker preventing and treating Using.

Background technology

Apple tree canker (Valsa mali Miyabe et Yamada) is commonly called as bark rot, smelly skin disease, is by black Beancurd sheet The important disease of shell category (Valsa ceratosperma) northern apple tree for causing.3 years knots of the raw above of the sick main harm Fruit tree, causes that tree vigo(u)r is weak, limb is withered, dead tree or even ruins garden.The disease symptoms are main based on ulcer type.Ulcer type is in early spring Occur bronzing, water stain shape, micro- protuberance, circle on trunk, bark to Long Circle scab, quality is soft, easily tear, hand pressure is easily recessed Fall into, flow out yellowish-brown juice, there is vinasse taste.Later stage drying shrinkage sink, and sick portion's edge crack has obvious pore i.e. conidium Device, pore is gushed out crocus tendril conidium when moist.Typically infect in 3-5 months, start morbidity the 7-8 months, early spring is hair Sick peak period.

Since the later stage forties 20th century, China's apple tree canker probably experienced 5 peak of occurrence.It is for the first time 1948-1951, the popular apple tree that result in up to a million plants is dead without restraint for South Liaoning apple tree canker.Occur for second In 1960-1962, due to the attention and control of all departments, the nineteen sixty-five state of an illness is controlled substantially.Third time occurs 1976 Before and after year, Bohai Sea Gulf old area, middle part ancient riverbed of the Yellow River area and the northwest plateau such as Shanxi, Shan, sweet area apple orchard are generally fallen ill, disease Feelings reach 15%-20%, and a small number of orchard diseased plant rates reach 70-80% and occur ruining garden phenomenon.It is popular in 1985-1986 4th time Spring, middle part fruit producing region and northwest fruit producing region to the west of the Zhengzhou of Bohai Sea Gulf old area, rot all universal generation (Zhao Xusheng, Qi Yong Will, Wang Yanan, Zhen Wenchao, Cao Ke wait by force nearly 50 years China's apple tree cankers a situation arises analysis, Guizhou Agricultural Sciences, 2014,42(11)130-132).It is very popular for 5th time and arises from 2005, Cao Ke waits by force national 10 province (city) 149 of investigation in 2008 Individual orchard finds that 4-24 apple tree canker diseased plant rates are up to 52%, if FUTURE ENVIRONMENT reaches the big occurrence condition of rot, The economic loss that cannot be estimated will certainly be brought to Chinese producing region.

The preventing and treating of apple tree canker is always based on chemical pesticide control, but chemical pesticide is long-term and substantial amounts of using Through causing environmental pollution, disruption of ecological balance, person poultry poisoning and killing the serious problems such as beneficial microbe.Current biological control is increasingly Approved by numerous researchers and orchard worker, therefore the exploitation biological control that safe, environment compatibility is good, validity is lasting Agent turns into the hot research direction of fungal diseases of plants integrated control.

At present, the disclosed biocontrol microorganisms and its microbial inoculum for preventing and treating apple tree canker are related to the more of bacterium and fungi：

The B of Chinese patent CN 104140938 disclose a kind of antagonistic bacterial strains for preventing and treating fruit tree putrefaction disease and its application.Should Invention Antagonistic Fungi is bacillus pumilus Bacilluspumilus XJAU-117, and microbial preservation number is CGMCCNo.9370. It is to separate to obtain from walnut rot in spite of illness branch, and effect, Ke Yiyou notable to the antagonism of the rot of fruit tree The growth of the suppression rot pathogen of effect, prevents and treats its plant disease for causing, and is that one plant of preventive effect is high, the good life of environmental safety Thing prevents and treats potentiality bacterial strain, with good development prospect.

The B of Chinese patent CN 103525708 disclose a kind of A.basellae and its in apple tree canker evil preventing and treating Application, the metabolic active substance of described A.basellae has compared with high inhibition effect to Pathogen of Apple Canker, can Rotten fungus mycelia deformity is caused, protoplast is excessive, and field control and therapeutic action show the culturing filtrate of the bacterial strain to apple Fruit tree putrefaction disease bacterium has good prevention effect.

The B of Chinese patent CN 104164394 disclose one plant of plant pathogenic fungi Antagonistic Fungi and its in controlling plant diseases In application.The invention Antagonistic Fungi is bacillus subtilis Bacillus subtilis ZSH-1, and microbial preservation number is CGMCC NO.9025.It is to separate to obtain from willow root soil, willow anthrax bacteria, Valsa sordida bacterium, willow can be prevented and treated Rotten pathogenic bacteria, Valsa mali, sweet Chinese bush cherry rotten pathogenic bacteria, Fusarium oxysporum and the microbial plant disease of jujube fruit-shrink disease, and The antibacterial volatile matter that the Antagonistic Fungi of the invention is produced can also prevent and treat willow anthrax bacteria, Valsa sordida bacterium, willow rotten pathogenic bacteria, Valsa mali, sweet Chinese bush cherry rotten pathogenic bacteria, Fusarium oxysporum and the microbial plant disease of jujube fruit-shrink disease.It is that a kind of preventive effect is high, Prevention and treatment range is wide, the good biological control potentiality bacterial strain of environmental safety, with good development prospect.

The activated species that biocontrol microorganisms disclosed above are produced are few, yield poorly, and activity is low, and the lasting period is short, protection effect It is undesirable.

Actinomyces are found from Cohn (1872) so far, it has been reported that 69 belong to 1687 kinds.In known actinomyces, half Tool Antagonism more than number is found, wherein mainly some species of streptomyces Streptomyces of biological control are applied to, Next has nocardia Nocardia, actinoplanes Actinoplanes, micromonospora Micromonospora etc..Nearly 10 Actinomyces produce and account for more than 70% in the new active substance found in Nian Laicong microorganisms, and the screening and application of biocontrol actinomycetes are Research emphasis as Plant Pathology field.

Actinomyces play Biocontrol Effect and typically pass through two kinds of approach to target bacterium：In host in vitro by antibiosis, competing The effect of striving, predation and hyperparasitism, causing the pathogenic and infect efficiency of pathogen reduces；Actinomyces also can be in host plant In tissue, induce it and produce resistance or produce antibacterial substance, influence growth, the breeding of pathogen, finally result in it dead.

Streptomycete is a kind of actinomyces, the antifungus active substance such as antibiotic, ectoenzyme that it is produced than probiotic bacteria and Activated species produced by fungi are more, active stronger, and the living cells preparation being made of its spore and mycelia is nontoxic, nothing Evil, noresidue, non-target microorganism is not injured and environment compatibility is good, the diseases prevention lasting period is long, and sporulation quantity is big, spore is degeneration-resistant Property it is strong, can be easily made living cells preparation such as spore pulvis, seed coat agent, Mycelium culture etc. and be easy to production and transportation to protect Deposit.It is widely used in the biological control of agricultural disease.

At present, the disclosed streptomycete for preventing and treating apple tree canker is less：

The A of Chinese patent CN 104673723 disclose a kind of streptomyces strain SL01 (Streptomyces extremely long Longissimus SL01) and the microbial bacterial agent (zymotic fluid and ferment filtrate) that is prepared by the bacterial strain.The invention is also disclosed The application of two kinds of microbial bacterial agents in apple tree canker is prevented and treated.Using different disposal method smear bacterial strain SL01 zymotic fluids and Ferment filtrate finds that the preventive effect of isolated shoot and fruit reaches 90%, and compareing thiophanate-methyl with medicament reaches same level； In field experiments in 2012, SL01 microbial inoculums prevention effect 100% is smeared after scraping scab, it is 58% that preventive effect is smeared after line；2013 In year field experiment, SL01 microbial inoculums prevention effect 97.8% is smeared after scraping scab, more than the preventive effect that thiophanate methyl medicament is compareed (84.6%) it is 64.4% that preventive effect, is smeared after line, can effectively be reduced to and prevent and treat apple tree canker and largely apply chemistry The destruction to ecological environment caused by agricultural chemicals, with good ecological and social benefit, development prospect is wide.

The B of Chinese patent CN 101822272 disclose a kind of application of Streptomyces Griseoflavus in biological control plant disease. Specially Streptomyces Griseoflavus (Streptomyces griseoflavus) NMG6-3-9 CGMCCNo.3441 are in biology for the application Application in controlling plant diseases.The bacterial strain has extraordinary antagonism to pathogens causing root rot disease of Medicago sativa, can be used in life Thing prevents and treats causing root rot disease of Medicago sativa and other plurality of plant diseases, is had broad application prospects in biological control plant disease field. The invention is laid the foundation for the biological control of causing root rot disease of Medicago sativa, and then development for the anti-Actinomycete preparation of the diease occurrence is provided with applying Scientific basis.

Zhang Qingming etc. have studied 5 karr streptomycete fermentation filtrates to the preventive effect of apple tree canker up to 70%.

Streptomycete protection effect disclosed above is not still highly desirable, and mechanisms of control wheat scab with most of bacteriums as fungi, Mainly by being metabolized the growth and breeding that produce the antibacterial materials such as antibiotic, antibacterial peptide to produce antagonistic effect suppression pathogen, Protection effect is not thorough, it is impossible to effect a radical cure apple tree canker.

The A of Chinese patent CN 105331545 disclose a kind of bacterial strain for preventing and treating apple tree canker, microbial bacterial agent and its Preparation method.The bacterial strain 61239 is preserved in China typical culture collection center on July 1st, 2015, and deposit number is CCTCC M2015420.The invention also includes microbial bacterial agent and its preparation of the chaetomium globosum containing apple tree canker is prevented and treated Method.In nature, pollution-free, nuisanceless, noresidue has no toxic side effect, either to apple biocontrol fungi to people and animals Fruit tree safety still all suitable to human body, ecological environment, can operate with any breeding time of apple tree, particularly rest period, Any resistance is not produced, can thoroughly control and effect a radical cure canker of apple fruit.It is demonstrated experimentally that to apple rot pathogen high specificity, Control effect is good, and average preventive effect is more than 95%.The bacterial strain 61239 has significant antibacterial and sterilization to Valsa mali Activity, particularly can with old on apple tree stick up skin, each cut saw kerf and put to death structural cellulose and lignin as nutrient, Long-term colonizing is survived on apple tree, can prevent the generation of new scab, and the recurrence of original old scar can be prevented again, can enduringly be controlled Valsa mali processed, so as to reach the purpose of thoroughly control and radical cure canker of apple fruit.

Although biocontrol microorganisms disclosed above can stick up skin with old on apple tree, each cuts saw kerf and put to death structural fiber Element and lignin be nutrient, colonize survive on apple tree for a long time, but can not directly degrade, using pathogen mycelium, with thorough Pathogen is killed at bottom, and biocontrol effect is poor.

Research shows：With chitin, cellulose as skeleton, with β -1,3- glucans and protein are the cell membrane of pathogen It is filled primarily with thing.Actinomyces are the main producing strains of antibiotic, and biocontrol actinomycetes suppress disease except the antagonism by antibiotic Outside opportunistic pathogen, in the presence of conventional inducer, the actinomyces pair of the extracellular hydrolases such as chitinase, β -1,3- dextranases are produced Pathogen has bacteriostasis, if can directly be grown with Pathogen of Apple Canker mycelium as nutrition, be bred, and Induction biocontrol microorganisms itself produce the extracellular hydrolase of various degradable pathogen cell membranes, and pathogen is made by the molten effect of coordinated enzyme Cell disruption, not only can directly kill pathogen, and can colonize for a long time at apple tree canker spot, thoroughly prevent apple tree The generation of rot, improves protection effect, effectively prevents rot from recurring.

To sum up, isolated one kind directly can be grown with Pathogen of Apple Canker mycelium as nutrition, it is numerous Grow, while induce itself producing the extracellular hydrolase of various degradable pathogen cell membranes to improve protection effect, prevent apple The streptomycete of tree rot recurrence is the expectation of those skilled in the art.

The content of the invention

Technical problem solved by the invention is to overcome existing apple tree canker biocontrol microorganisms --- streptomycete mechanisms of control wheat scab Defect, be research object with microbial streptomycete characteristic, it is special with Pathogen of Apple Canker pathological characteristics, trophic structure Property and extracellular hydrolase metabolic characteristic for screening target, separate, screening one plant can be directly with Pathogen of Apple Canker Mycelium is grown for nutrition, is bred, while can induce itself the extracellular water of various degradable pathogen cell membranes is produced Solution enzyme, colonizes at apple tree canker scab for a long time, to improve protection effect, the strepto- for preventing apple tree canker from recurring Bacterium.

Primary and foremost purpose of the present invention is to provide one plant of Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin- 1。

In order to achieve the above object, the present invention uses following technical scheme：

Separation, primary dcreening operation, purifying obtain 12 plants and have substantially suppress apple tree to rot from the orchard soil of Yantai City The streptomycete of sick pathogen, then secondary screening obtain 8 plants higher to Pathogen of Apple Canker inhibiting rate of streptomycete, and through producing Chitinase Screening of Media obtains 6 plants and produces the stronger streptomycete of chitinase ability, then is obtained through base agar Screening of Media 5 plants with Pathogen of Apple Canker mycelium as nutrient growth, breeding streptomycete, most afterwards through extracellular hydrolase (chitin Matter enzyme, β -1,3- dextranases, polyphenol oxidase, protease) screening active ingredients, 1 plant is finally given to apple tree canker cause of disease Bacterium inhibiting rate highest, and using pathogen mycelial growth, breed and induce itself produce various degradable pathogen cell membranes, The streptomycete Actin-1 of activity extracellular hydrolase higher, comprehensive morphological characteristic, cultural character, physio-biochemical characteristics, heredity are special Property is accredited as Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1.

Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1 be from Yantai City really Separated in Orchard Soil, purified, screening is obtained.The bacterial strain is preserved in Chinese microorganism strain preservation on March 24th, 2017 (abbreviation CGMCC, address is administration committee's common micro-organisms center：Section of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Institute of microbiology of institute, postcode：100101), deposit number is CGMCC NO.13927, and Classification And Nomenclature is micro- Bai Huanglian Mould Streptomyces albidoflavus.

Streptomycesalbidoflhaving (Streptomyces albidoflavus) the Actin-1 strain classifications status is bacterium Door, Actinomycetes, Actinomycetal, Actinomy cetaceae, streptomyces, growth temperature range are 15-45 DEG C, and optimum growth temperature is 30 ℃；PH tolerant scope is 3.0-10.0, and the most suitable growth pH value is 7.0；The sodium chloride solution of tolerable 1-10%, grows good It is good, grown when concentration is 3-5% best；Can be grown on various culture mediums.

Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1 is to apple tree canker cause of disease The bacteriostasis rate of bacterium is 89.82%；To Botryosphaeria berengeriana, rod method apple specialized form, Phytophthora cactorum, cherry alternaric bacteria, Various pathogens such as verticillium dahliae, cherry spherical cavity bacterium, Rhizoctonia solani Kuhn, Botrytis cinerea, anthrax bacteria, apple rod method With preferable fungistatic effect, bacteriostasis rate is 76.08-87.10%, with broad-spectrum antibacterial；

Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1 is with apple tree canker disease Opportunistic pathogen mycelium for sole carbon source basal medium (liquid and agar) in can normal growth, breeding, with following characteristic：

1) induce and itself produce various extracellular hydrolases, wherein chitinase activity peak value is 140.26U/mL；β-1,3- Dextranase activity peak value is 49.46U/mL；Polyphenol oxidase activity peak value is 39.25U/mL；Proteinase activity peak value is 10.90U/mL；

2) spore germination to Pathogen of Apple Canker has inhibitory action：Sterilized water control treatment, apple tree is rotted Sick pathogen spore germination rate is 98.60%, and adds Streptomycesalbidoflhaving crude enzyme liquid to process, Pathogen of Apple Canker spore Sub- germination rate is only 3.60%, and relatively control inhibiting rate improves 96.35%；

3) there is obvious dissolution to Pathogen of Apple Canker mycelium：Compare with sterilized water, crude enzyme liquid during 72h The pathogen aerial mycelium for the treatment of is sunk, and bacteriolyze loop diameter produces obvious dissolution up to 6mm.Proteinase K dissolution Most significantly, dissolving loop diameter is 10mm.And myceliolysis does not occur in sterilized water control CK, the pressure of water droplet effect is only shown Trace；

4) there is significant fungistatic effect to Pathogen of Apple Canker：The stronger antibacterial substance of activity can be produced, it is antibacterial Rate is up to 96.7%.

After Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1 is through 30 generations of biography, to apple tree The bacteriostatic activity of rot pathogen and other pathogens is basically identical with Streptomycesalbidoflhaving Actin-1 1st generation bacteriostasis rates, table Bright Streptomycesalbidoflhaving Actin-1 of the present invention will not reduce inhibitory activity with the increase of passage number, with higher Genetic stability.

Another object of the present invention is to provide above-mentioned Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin- 1 application in terms of apple tree canker preventing and treating.

Tested through the Curettage for treating of field apple tree canker scab, result of the test shows Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1 can effectively prevent apple tree canker from recurring, and prevention effect is 100%, Recurrence rate is 0, and can be colonized for a long time at the apple tree canker scab cured, and plays long-term biological control and makees With.

Beneficial effect：

1. Streptomycesalbidoflhaving Actin-1 of the present invention can be given birth to by nutrition of Pathogen of Apple Canker mycelium Long, breeding, colonizes for a long time beneficial to biocontrol microorganisms at apple tree canker spot, long-term biological control effect is played, while induction Itself produces various extracellular cytohydrolists, and pathogen cell disruption is made by the molten effect of coordinated enzyme, and can produce compared with Strong antibacterial substance, produces inhibitory action, number of mechanisms collective effect to prevent apple Pathogen of Apple Canker growth The growth of fruit tree putrefaction disease pathogen, breeding, substantially increase the biocontrol effect to apple tree canker.

2. Streptomycesalbidoflhaving Actin-1 of the present invention is 89.82% to the bacteriostasis rate of Pathogen of Apple Canker；To shellfish It is human relations lattice grape seat chamber bacterium, rod method apple specialized form, Phytophthora cactorum, cherry alternaric bacteria, verticillium dahliae, cherry spherical cavity bacterium, vertical Various pathogens such as withered silk kernel fungus, Botrytis cinerea, anthrax bacteria, apple rod method have preferable fungistatic effect, antibacterial Rate is 76.08-87.10%, with broad-spectrum antibacterial；

3. Streptomycesalbidoflhaving Actin-1 tolerable temperatures of the present invention are high, and good life can be kept under conditions of 45 DEG C Long status, illustrate that Streptomycesalbidoflhaving Actin-1 can not only keep stronger spawn activity during the fermentation, on the other hand Can also illustrate, its biocontrol agent can keep notable during the preventing and treating for being applied to pathogen, not only under normal temperature condition Fungistatic effect, and good bacterial strain vigor and inhibitory effect can be remained in that in hot weather, with stronger resistance to Hou Xing.

4. Streptomycesalbidoflhaving Actin-1 of the present invention has acid resistance and alkali resistance, in the range of pH value 3.0-10 soda acids, Still good growth conditions are presented, stronger spawn activity can be not only kept during the fermentation, in biocontrol agent application During the preventing and treating of pathogen, it is also possible to the extraneous different acid or alkali environment of resistance, while Antibacterial Activity higher is kept, it is acidproof It is alkaline strong.

5. after Streptomycesalbidoflhaving Actin-1 of the present invention is through 30 generations of biography, to Pathogen of Apple Canker and other cause of diseases The bacteriostatic activity of bacterium is basically identical with Streptomycesalbidoflhaving Actin-1 1st generation bacteriostasis rates, shows Streptomycesalbidoflhaving Actin-1 Inhibitory activity will not be reduced with the increase of passage number, with genetic stability higher.

6. Streptomycesalbidoflhaving Actin-1 fermentative activities of the present invention are high, by liquid fermentation seed liquor and solid fermentation and It is 6.90 × 10 that the selection of auxiliary agent finally obtains bacterium vigor¹²The biocontrol agent of cfu/g, prevents and treats effect in apple tree canker application Up to 100%, recurrence rate is zero to fruit, while the bacterium may additionally facilitate the healing of scab wound callus.Separating experiment shows micro- again Yellow streptomycete Actin-1 successes go out in apple tree canker scab and colonize for a long time in vain, and it is very high to colonize rate.

Therefore, it is main biocontrol microorganisms with the bacterium, carries out preventing and treating the fruits and vegetables pathogen such as apple tree canker, with prevention effect Good (bacteriostasis rate is high), efficiency high (thoroughly degraded pathogen cell), recurrence rate low (colonization ability is strong at scab), adaptation environment energy Power strong (high temperature resistant, resistance to acids and bases are strong), stabilization strong (genetic stability is strong), the multiple advantage such as the resistance to the action of a drug is not likely to produce, is biological The first-selection of preventing and treating, can research and develop the biological and ecological methods to prevent plant disease, pests, and erosion products such as various biocontrol agents, biological and ecological methods to prevent plant disease, pests, and erosion nutritional agents, biological pesticide, biocontrol bacterial fertilizer, to carrying The prevention effect of the fruits and vegetables pathogen such as apple tree canker high, prevent pathogen recur, environmental protection it is significant.

Brief description of the drawings

The step 3 of Fig. 1 embodiments 1) face-off experiment one bacteriostasis rate screening inoculation schematic diagram；

The step 6 of Fig. 2 embodiments 1) 5 streptomycetes with apple tree pathogen mycelium be sole carbon source produce chitin enzyme activity Proterties condition；

The step 6 of Fig. 3 embodiments 1) 3 streptomycetes produce β -1,3- Portugals to gather by sole carbon source of apple tree pathogen mycelium Anase activity situation；

The step 6 of Fig. 4 embodiments 1) 5 streptomycetes with apple tree pathogen mycelium be sole carbon source produce polyphenol oxidase Active situation；

The step 6 of Fig. 5 embodiments 1) 3 streptomycetes with apple tree pathogen mycelium be sole carbon source produce proteinase activity Situation；

The step 7 of Fig. 6 embodiments 1) streptomycete Actin-1 colony growth features on Gause I agar medium；

The step 7 of Fig. 7 embodiments 1) streptomycete Actin-1 mycelia and spore chain form under an optical microscope；

The step 7 of Fig. 8 embodiments 1) streptomycete Actin-1 and strepto- based on 16S rDNA belong to the systematic growth of related strain Tree；

The step 7 of Fig. 9 embodiments 1) streptomycete Actin-1 based on gyrB and strepto- belong to the phylogenetic tree of related strain；

The normal mycelia microscopy figure of the control group of Figure 10 embodiments 4；

The treatment group of Figure 11 embodiments 4 variation mycelia microscopy figure；

The Streptomycesalbidoflhaving Actin-1 biocontrol agents of Figure 12 embodiments 9 cure apple of the apple tree canker scab after 4 years Tree rot scab healing effect figure.

Specific 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 On the premise of invention spirit and scope, the various changes that are carried out to the material component and consumption in these embodiments or change Belong to protection scope of the present invention.

The separation of the Streptomycesalbidoflhaving of embodiment 1 (Streptomyces albidoflavus) Actin-1, screening and mirror It is fixed

1) separation, primary dcreening operation：10g Yantai Cities orchard soil sample accurately is weighed, is added to aseptic containing 100mL coolings In the band bead sterilizing triangular flask of water, the concussion of 200r/min shaking tables mixes 30min, stands 5min Aspirate supernatant 1mL, successively Gradient dilution obtains 10^-3~10^-9Concentration, chooses 10^-3、10^-4、10^-5、10^-6、10^-7Diluted concentration, drawn respectively with pipettor Each gradient suspension 100 μ L, with spreader even spread to screening and culturing medium flat board, are compareed, by flat-plate inverted with sterilized water Incubator is placed in, 30 DEG C are cultivated 7 days, each treatment setting level repeats 3 groups, according to transparent circle size and microbe colony Shape, size, produce spore situation, surface texture, marginal texture, quality, color or generation can the aspect such as lysochrome feature, just Step screening obtains 12 plants has the streptomycete for substantially suppressing Pathogen of Apple Canker；

The preparation method of the screening and culturing medium flat board is：Gause I agar medium after subject to sterilization is cooled to 55- At 60 DEG C, the concentration of filtration sterilization is added thereto to for 15mg/mL nalidixic acid solution, addition is every 300mL additions 10mL, Uniform mixing；Continue to the Pathogen of Apple Canker spore suspension that 10% (V/V) is added in mixed culture medium, mixing Uniformly, flat board is made to obtain final product；

The Gause I agar medium quality group turns into：Soluble starch：20g, NaCl 0.5g, KNO₃：1g, K₂HPO₄：0.5g, MgSO₄·7H₂O：0.5g, FeSO₄·7H₂O：0.01g, agar 20：G, distilled water：1L, pH value 7.2-7.4；

The preparation method of the Pathogen of Apple Canker spore suspension is：Pathogen of Apple Canker point is connected to PDA culture medium flat board, 28 DEG C are cultivated 7 days, with aseptic water elution, scraping spore and mycelia in the triangular flask equipped with stirrer, The suspension of spore containing Pathogen of Apple Canker and mycelia is obtained, concussion, magnetic agitation make spore fully dispersed, be filtered to remove Mycelia obtains spore suspension, and being diluted with sterilized water makes spore content be 1 × 10⁷Individual/mL is obtained final product；

The PDA culture medium quality group turns into：Potato (200g) filtrate：1L, glucose：20g, agar：20g, pH value It is natural；

2) purify：By step 1) obtain 12 streptomycetes connect in the flat lining out of Gause I agar medium respectively Kind, 30 DEG C are cultivated 7 days according to upgrowth situation, and selection colony diameter is larger, and the bacterium colony of robust growth is screened, purified, so Repeatedly, until being considered as the bacterium colony of single culture when the growth conditions of bacterium colony show as consistent with morphological feature, 12 plants are so obtained There is the purifying streptomycete of obvious fungistatic effect to Pathogen of Apple Canker, numbering is Actin-0 to Actin-11；

3) secondary screening (face-off experiment one)：By step 2) obtain 12 plants purifying streptomycete streak inoculations in Gause I agar Culture medium flat plate, 30 DEG C of cultures obtain activation streptomycete for 7 days；Pathogen of Apple Canker point is connected to PDA culture medium flat board, 28 DEG C of cultures obtain activation pathogen for 7 days；Crossed at the PDA culture medium flat board back side of diameter 90mm, be with right-angled intersection point The flat board center of circle, 4 points are inoculated with 4 activation streptomycete bacteria cakes of 6mm respectively at the 25mm of the center of circle on cross hairs, are connect in circle centre position 1 activation pathogen bacteria cake (such as Fig. 1) of 6mm is planted, as treatment group；Only inoculation activation pathogen bacteria cake is not inoculated with activation strepto- Bacterium bacteria cake is as a control group；It is respectively placed in 28 DEG C to cultivate 7 days, control pathogen radius and treatment pathogen half is counted respectively Footpath, calculates bacteriostasis rate, the results are shown in Table 1, chooses 8 plants of bacteriostasis rates streptomycete higher：Actin-0、Actin-1、Actin-3、 Actin-6、Actin-7、Actin-9、Actin-10、Actin-11；

Bacteriostasis rate (%)=(control pathogen radius mm- treatment pathogens radius mm)/control pathogen radius mm × 100；

Table 1：Bacteriostasis rate of 12 plants of purifying streptomycetes to Pathogen of Apple Canker

4) chitinase primary dcreening operation：By step 3) the bacteriostatic activity 8 streptomycete points higher that obtain are connected to chitinase mirror Other culture medium flat plate, the flat board of bacterium is not connect as control, 3 repetitions of every plant of bacterium, after 30 DEG C of constant incubator lucifuges culture 7 days, 0.2g/L congo red stainings, 1mol/LNaCl is used to decolourize, observation hydrolysis circle whether there is, and bacterium colony and hydrolytic circle is recorded, with hydrolysis The ratio evaluation of loop diameter/colony diameter produces chitinase activity just, final to obtain 6 plants of product chitinase energy as a result such as table 2 The stronger streptomycete of power：Actin-0、Actin-1、Actin-3、Actin-7、Actin-10、Actin-11；

The chitinase differential medium quality composition：Tobacco brown spot pathogen：15.0g, KH₂PO₄：0.7g,MgSO₄· 7H₂O：0.5g, FeSO₄·7H₂O：0.01mg, ZnSO₄：0.001g, distilled water 1L, agar 20g, pH value nature；

Table 2：8 plants of purifying streptomycete Actin-1 produce chitinase ability

5) pathogen mycelium utilizes secondary screening：By step 4) obtain 6 plants product chitinase streptomycete streak inoculations in On base agar culture medium flat plate with Pathogen of Apple Canker mycelium as sole carbon source, 3 repetitions of every plant of bacterium, 30 DEG C After constant incubator lucifuge culture 7 days, the results are shown in Table 3, choose 5 plants can normal growth, the streptomycete Actin-1 of breeding, Actin-3、Actin-7、Actin-10、Actin-11；

The base agar culture medium quality is constituted：Pathogen of Apple Canker mycelium：10g, K₂HPO₄· 3H₂O：0.5g, KNO₃：1g, MgSO₄·7H₂O：0.5g, FeSO₄·7H₂O：10mg, distilled water：1L, agar：20g, pH value is certainly So；

The mycelial preparation method of Pathogen of Apple Canker is：Picking step 3) the activation pathogen bacterium Silk, is inoculated into the 500mL shaking flasks equipped with 100mL Cha Shi fluid nutrient mediums, 30 DEG C, 160rpm shaking tables shaken cultivation 10 days.With The double gauze of sterilizing is collected by filtration mycelium, with deionized water rinsing 5-10 times to noresidue nutrient solution, 45 DEG C of drying, grinding To fine-powdered, cross 60 mesh sieves and obtain final product；

The Cha Shi fluid nutrient mediums quality composition：NaN0₃：3g, K₂HPO₄：1g, MgSO₄·7H₂O：0.5g, KCl： 0.5g, FeSO₄·7H₂O：0.01g, sucrose：30g, distilled water：1L, pH value nature；

Table 3：Streptomycesalbidoflhaving Actin-1 utilizes pathogen mycelia ability

6) Extracellular enzyme activity secondary screening is produced：By step 5) obtain 5 streptomycetes carry out respectively Liquid Culture and prepare slightly Enzyme liquid, determine respectively its produce extracellular chitinase (Chitinase), β -1,3- dextranases (β -1,3-glucosidase), The activity of polyphenol oxidase (PPO) and protease (Protease), the results are shown in Table 4, chooses and produces Extracellular enzyme activity highest bacterium Strain Actin-1 is final bacterium；

The preparation method of the crude enzyme liquid is：By 5 streptomycete Actin-1, Actin-3, Actin-7, Actin-10, Actin-11 is activated respectively, the same step 3) of activation method, and every plant of activation streptomycete is inoculated with a ring to the basis equipped with 100mL respectively In the 500mL triangular flasks of fluid nutrient medium A, 3 repetitions of every plant of bacterium, 30 DEG C, 160rpm shaking table cultures, the 3rd, 5,7 and 9 days in 20mL nutrient solutions are suctioned out with aseptic straw on super-clean bench, a small amount of sterile absorbent cotton is placed in funnel bottom filtering spore, filtrate is in 4 000rpm is centrifuged 5min, and supernatant is crude enzyme liquid；

The basal liquid medium A quality groups turn into：Pathogen of Apple Canker mycelium：10g, K₂HPO₄· 3H₂O：0.5g, KNO₃：1g, MgSO₄·7H₂O：0.5g, FeSO₄·7H₂O：10mg, distilled water：1L, pH value nature；

A. chitinase activity is determined：Take 1mL crude enzyme liquids, plus 1mL 10g/L tobacco brown spot pathogen, 37 DEG C are placed in after mixing 3h is hydrolyzed in water bath with thermostatic control, 5min terminating reactions are boiled in boiling water bath, reaction solution is centrifuged 5min, takes 1mL supernatants in 4 000rpm Or dilution DNS methods determine 2-Acetamido-2-deoxy-D-glucose content in hydrolyzate.

1mL enzyme liquids at 37 DEG C are hydrolyzed into the chitinase activity that chitin produces 1 μ g 2-Acetamido-2-deoxy-D-glucoses in 1h It is defined as 1U.

As a result：When adding Pathogen of Apple Canker mycelium as the sole carbon energy in basal liquid medium A, Chitinase activity, but enzyme activity can be detected in Actin-1, Actin-3, Actin-7, Actin-10, Actin-11 nutrient solution Not of uniform size, Actin-1 chitin enzyme activity peak values are up to 140.26U/mL, and Actin-10 enzyme activity peak values are minimum, are 62.45U/ mL.The chitinase activity of different incubation time crude enzyme liquids is different, increases with incubation time, and chitinase activity increases, Actin-1, Actin-3 and Actin-11 the 5th day, enzyme activity peaking, at the 7th day, enzyme activity reached peak to Actin-7 and Actin-10 Value, up to enzymatic activity peak value after with incubation time increase enzymatic activity reduction, see Fig. 2；

B. beta-1,3-glucanase determination of activity：0.5mL crude enzyme liquids are taken, the laminarin of 0.5mL 10g/L is added, mixed After be placed in 45 DEG C of waters bath with thermostatic control and hydrolyze 30min.Hydrolyzate is placed in 5min terminating reactions in boiling water bath.Take 1mL hydrolyzates or Dilution DNS methods determine glucose content in hydrolyzate.

By 1mL enzyme liquids at 45 DEG C, hydrolyses laminarin produces the beta-1,3-glucanase activity of 1 μ g glucose in 1min It is defined as 1U.

As a result：When adding Pathogen of Apple Canker mycelium as the sole carbon energy in basal liquid medium A, β -1,3- dextranase activities can be detected in Actin-1, Actin-3 and Actin-11 nutrient solution, and enzyme activity reached at the 7th day To peak value and enzyme activity is higher, respectively 49.46U/mL, 42.38U/mL, 16.24U/mL, Actin-7 and Actin-10 nutrient solutions In cannot not detect beta-1,3-glucanase activity.See Fig. 3.

C. polyphenol oxidase activity is determined：The acetate for taking catechol solution 3mL, 0.1mol/L of 0.02mol/L delays Fliud flushing (pH value 4.8) 3mL, crude enzyme liquid 1mL are added in test tube, are fully shaken up, and 10min is hydrolyzed in 30 DEG C of waters bath with thermostatic control, Mensuration absorbance value under 400nm wavelength, 1U is defined as with every point of OD value changes 0.01；

As a result：When in basal liquid medium A using Pathogen of Apple Canker mycelium as the sole carbon energy, Polyphenol oxidase activity can be detected in Actin-1, Actin-3 and Actin-7, Actin-10, Actin-11 crude enzyme liquid, Actin-1 and Actin-7 enzyme activity is higher, and increasing with incubation time and increasing, enzymatic activity peaking at the 7th day, respectively 39.25U/mL and 31.42U/mL, enzyme activity reduction is increased after peaking with incubation time.Actin-3, Actin-10 and Actin- 11 producing enzyme work are relatively low, see Fig. 4.

D. protease activity is determined：40 DEG C of 1min caseinhydrolysates of 1mL crude enzyme liquids are produced 1 μ g tyrosine by Folin reagent method Proteinase activity be defined as 1U.

As a result：When adding Pathogen of Apple Canker mycelium as the sole carbon nitrogen energy in basal medium A Proteinase activity can be detected in Actin-1, Actin-10, Actin-11 nutrient solution, and peak value is higher, respectively：10.9U/mL、 9.8U/mL, 10.2U/mL, proteinase activity is not detected in Actin-3 and Actin-7 nutrient solutions.Show apple tree canker disease Albumen enzymatic synthesis of the opportunistic pathogen mycelium to Actin-1, Actin-10, Actin-11 has inducing action.See Fig. 5.

Table 4：54 kinds of streptomycete different times hydrolytic enzyme activities (U/mL)

Therefore, finally filter out bacteriostasis rate it is high, can with Pathogen of Apple Canker mycelium as sole carbon source growth, Breed, produce various extracellular hydrolases and enzymatic activity strain streptomycete Actin-1 higher, slant strains preservation is standby.

7) strain idenfication

Streptomycete Actin-1 is identified according to morphology, Physiology and biochemistry and heredity feature, is comprised the following steps that：

(1) Morphology level identification

Standard medium (the Shiring and of defined are described using international streptomycete plan actinomyces cultural characteristic Gottlieb 1996)) Gause I agar medium, oatmeal agar medium, brewer's wort yeast extract agar culture medium, nothing Machine salt starch agar medium, glycerine asparagine agar medium, sucrose Cha Shi agar mediums, glucose yeast cream agar Streptomycete Actin-1 slant strains are distinguished the above-mentioned culture medium of streak inoculation by culture medium etc., and after plate seal, 30 DEG C are inverted training Support 8-15 days.Observational record colonial morphology, the positive and negative colour consistency of bacterium colony, the dense degree of mycelial growth, and whether produce can Dissolubility pigment and pigmentary colours, cultural characteristic describe reference《Actinomyces Rapid identification and genealogical classification》(Ruan Jisheng, Huang Ying etc.), 《Streptomycete identification handbook》(Institute of Microorganism, Academia Sinica actinomyces separation group 1975) is recorded, as a result such as table 5.

The positive and negative color of streptomycete Actin-1 is inconsistent on Gause I agar medium, and front is taupe, reverse side It is chestnut brown, sporulation quantity is big, and bacterium colony quality consolidation is fine and close, produces amber water colo(u)r, sees Fig. 6, the gentle life of substrate mycelium Mycelial growth is in great numbers, and the ability that substrate mycelium is attached to culture medium is strong, and aerial hyphae is spirality, sees Fig. 7.

Cultural characteristic on the streptomycete Actin-1 different culture medias of table 5

Note：Result is defined by last time observation result；

The Gause I agar medium quality composition：Soluble starch：20g, K₂HPO₄·3H₂O：0.5g, NaCl： 0.5g, KNO₃：1g, MgSO₄·7H₂O：0.5g, FeSO₄·7H₂O：0.01g, distilled water：1L, agar：20g, pH value 7.2-7.4；

Brewer's wort yeast extract agar culture medium (ISP2) quality is constituted：Dusty yeast:4g, malt flour:10g, glucose:4g, Agar 20g, distilled water：1L, pH value 7.2-7.3；

Oatmeal agar medium (ISP3) the quality composition：Oatmeal:20g, agar:20g, Trace salts solution: 1mL, distilled water is supplemented to 1L, pH value 7.2；

The inorganic salts starch agar medium (ISP4) constitutes：Soluble starch:10g, K₂HPO₄:1g, MgSO₄· 7H₂O:1g, NaCl:1g, (NA₄)₂SO₄:2g, CaCO₃:2g, Trace salts solution 1mL, agar:20g, distilled water：1L, pH value 7.0- 7.4；

Glycerine asparagine agar medium (ISP5) the quality composition：Glycerine：10g, altheine：1g, it is micro Salting liquid：1mL, K₂HPO₄：1g, agar：20g, distilled water 1L, pH value 7.0-7.4；

The sucrose Cha Shi agar mediums quality composition：NaN0₃：2g, K₂HPO₄：1g, MgSO₄·7H₂O：0.5g, KCl: 0.5g, FeSO₄·7H₂O：0.01g, sucrose：30g, distilled water：1L, agar：20g, pH value nature；

The glucose yeast cream agar medium quality composition：Yeast extract：10g, glucose：20g, distilled water：1L, fine jade Fat：20g, pH value 7.2-7.4；

The Trace salts solution quality composition：FeSO₄·7H₂O：0.1g, MnCl₂·4H₂O：0.1g, ZnSO₄·7H₂O： 0.1g, distilled water：100mL；

(2) physio-biochemical characteristics

The streptomycete Actin-1 catalases experiment, lipase experiment, amylase experiment, hydrolysis xanthine degraded Experiment, gelatin liquefaction experiment, nitrate reductase, amicillin resistance experiment, cellulose hydrolysis, Starch Hydrolysis experiment are sun Property；Oxidizing ferment generation experiment, melanin generation are tested, hydrogen sulfide produces experiment, casein degradation experiment, aesculin utilize experiment, Rifampicin resistance experiment is feminine gender；Thalline can using D-Glucose, L (+)-rhamnose, sucrose, D-MANNOSE, L- I Uncle's sugar, lactose, sorbierite, xylose, D- galactolipins, sodium acetate, salicin, cellobiose, altheine, it is impossible to using horse urine Sour sodium, can grow under 1-10% scope salt concentration conditions, and growing way is best in the range of 3-5%；

Reference《Actinomyces Rapid identification and genealogical classification》The yellow English of Ruan Ji lifes write and《The outstanding Bacteria Identification handbook the (the 8th of uncle Version)》Related content carry out Physiology and biochemistry identification.Major physiological biochemical characteristic such as table 6.

The streptomycete Actin-1 major physiological biochemical characteristic result of the tests of table 6

Note："+" represents the positive, and "-" represents feminine gender, and in utilization of carbon source experiment, "+" represents growth, and "-" is represented and do not given birth to Long, " ++ " represents well-grown, and " +++ " represents riotous growth.

(3) hereditary capacity：

Gene is extracted：Streptomycete Actin-1 is inoculated in 2216E fluid nutrient mediums, 30 DEG C of concussion and cultivates 3 days, to training Occur cenobium in nutrient solution, cenobium is added to centrifuge tube (1.5mL), gene is extracted in strict accordance with extracting genomic kit flow Group；

It is template with the DNA genomes for extracting, using 27f/1492r (27f：5′-GAGTTTGATCCTGGCTCAG-3′； 1492r：5 '-ACGGCTACCTTGTTACGACTT-3 ') expand 16S DNA (16s rDNAs) for primer enters performing PCR reaction Sequence fragment, PCR reaction conditions：94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 40s, 72 DEG C of 1.5min, 30 circulations, 72 DEG C extend eventually 10min.Simultaneously using gyrBPF/gyrBPR (gyrBPF:5′- GAGGTCGTGCTGACCGTGCTGCACGCGGGCGGCAAGTTCGGC-3′；gyrBPR:5′- GTTGATGTGCTGGCCGTCGACGTCGGCGTCCGCCAT-3 ') expand gyrB (gyrase (gyrase) B for primer enters performing PCR Subunit gene) sequence fragment, PCR reaction conditions：95 DEG C of 5min, 95 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 1min, 30 circulations, 72 DEG C eventually extend 10min.(gel extraction step is in strict accordance with full formula gold size QIAquick Gel Extraction Kit side for gel extraction PCR primer after electrophoresis Method is operated), pEASY T3 carriers are connected, and then using thermal shock conversion method conversion Top competence, blue hickie screens picking hickie Bacterium colony is added into the LB culture mediums for adding Amp, 37 DEG C, 200rpm cultures 8-12h.Positive colony is carried out using T7/SP6 primers Detection, delivers to positive colony bacterium solution Beijing Liuhe Huada Genomics Technology Co., Ltd and is sequenced, the sequence that will be obtained After row splicing check and correction, carry out Blast using GeneBank databases and compare analysis, Clustal X2.0 softwares carry out homology point Analysis, the Neighbor-Joining method phylogenetic tree constructions in MEGA7.0 softwares determine strain kind.

The 2216E fluid nutrient mediums quality composition：Bacto peptone：5g, dusty yeast：1g, distilled water：1L, pH value is certainly So；

Molecular Identification result is as follows：

Final 16S rDNA measure sequence length for 1502bp (see sequence table), and gyrB measures sequence length for 1286bp (see sequence table)；Show the 16S rDNA sequences and Streptomycesalbidoflhaving of streptomycete Actin-1 through EzbioCloud comparison results (Streptomyces albidoflavus DSM 40455 (T)) is up to 99.66% similitude, sees Fig. 8；Streptomycete The gyrB gene orders of Actin-1 compare analysis and Streptomycesalbidoflhaving (Streptomyces through NCBI Blast Albidoflavus NRRLB-1271) 99% similitude is up to, see Fig. 9；And two kinds of systems of gene of streptomycete Actin-1 Phylogenetic analysis result identifies that Actin-1 is Streptomycesalbidoflhaving.

In sum, it is last true according to morphological character, cultural character, physio-biochemical characteristics and hereditary capacity comprehensive analysis Streptomycete Actin-1 is determined for Streptomycesalbidoflhaving (Streptomyces albidoflavus).

The Streptomycesalbidoflhaving Actin-1 growth temperatures of embodiment 2 and pH resistance test

The preparation of Streptomycesalbidoflhaving Actin-1 spore suspensions：By Streptomycesalbidoflhaving Actin-1 streak inoculations in Gao Shi On a number agar medium inclined-plane, 30 DEG C of cultures extremely produce spore in 7 days；5mL sterilized waters are added, spore is scraped into nothing with disinfection inoculation pin In bacterium water, concussion is mixed, and adjustment spore concentration is 1 × 10⁶Individual/mL obtains spore suspension.

1. growth temperature experiment

2216E fluid nutrient mediums are prepared, is sub-packed in 48 500mL triangular flasks, 100mL/ bottles, 121 DEG C of sterilizing 30min, It is standby；Above-mentioned spore suspension is inoculated in 36 triangular flasks therein according to 5 ‰ (V/V), as treatment group, 12 not be inoculated with Individual triangular flask as a control group, according to 0 DEG C, 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C of thermograde packets, 1 control of every group of 3 treatment carries out incubated, condition of culture 200rpm concussion and cultivates 5 days, sees Examine and record the growing state of Streptomycesalbidoflhaving Actin-1 at each temperature.The results are shown in Table 7.

2. resistance to acids and bases experiment

With dilute HCl or NaOH solution by 2216E fluid nutrient mediums be adjusted to different pH value (1.0,2.0,3.0,4.0,5.0, 6.0th, 7.0,8.0,9.0,10.0,11.0,12.0), each pH value 2216E fluid nutrient mediums are dispensed in 4 500mL triangular flasks, 100mL/ bottles, sterilize 30min in 121 DEG C.Above-mentioned spore suspension is inoculated into respectively by 5 ‰ (V/V) above-mentioned equipped with different pH value 2216E fluid nutrient mediums in.Each pH value fluid nutrient medium is inoculated with 3, is set to treatment group, and 1 is not inoculated with, and is set to control Group, all in 30 DEG C, 200rpm concussion and cultivates 5 days record Streptomycesalbidoflhaving Actin-1 growing states under each pH value.As a result It is shown in Table 7.

The Streptomycesalbidoflhaving Actin-1 growth temperatures of table 7 and acid and alkali-resistance result

Note："+" represents growth, and "-" is represented and do not grown, and " ++ " represents well-grown, and " +++ " represents riotous growth.

As can be seen from Table 7, Streptomycesalbidoflhaving Actin-1 has stronger heat resistance, can be given birth in 45 DEG C of condition It is long, thus illustrate, Streptomycesalbidoflhaving Actin-1 can not only keep significant fungistatic effect, Er Qieke under normal temperature condition In the weather of high temperature, to remain in that good bacterial strain vigor and inhibitory effect.

Streptomycesalbidoflhaving Actin-1 has acid resistance and extremely strong alkali resistance, is coerced in the soda acid of pH value 3.0-10.0 Under compeling, can grow, illustrate that Streptomycesalbidoflhaving Actin-1 can not only keep stronger spawn activity during the fermentation, And during the preventing and treating that biocontrol agent is applied to pathogen, it is also possible to the extraneous different acid or alkali environment of resistance, so as to keep Antibacterial Activity higher.

The Streptomycesalbidoflhaving Actin-1 broad-spectrum antibacterials of embodiment 3 are verified and genetic stability experiment：

Streptomycesalbidoflhaving Actin-1 is carried out to Pathogen of Apple Canker (apple black skin shell bacterium), apple spot The bacteriostatic experiment of the 11 pathogen strain bacterium such as defoliation pathogen (rod method apple specialized form), concrete operation step is as follows：

1) Streptomycesalbidoflhaving Actin-1 streak inoculations are cultivated 5 in Gause I agar medium flat board in 30 DEG C My god；Pathogen of Apple Canker point is connected to PDA culture medium flat board, is cultivated 7 days in 28 DEG C；Obtain activation Streptomycesalbidoflhaving Actin-1 and activation pathogen；

2) face-off experiment two：Crossed at the PDA culture medium flat board back side of diameter 90mm, justified by flat board of right-angled intersection point The heart, 3 points are inoculated with 3 activation Streptomycesalbidoflhaving Actin-1 bacteria cake conducts of 6mm respectively at the 25mm of the center of circle on cross hairs Treatment group, the 4th point does not connect bacterium as a control group；1 activation pathogen bacteria cake of 6mm is inoculated with circle centre position；It is respectively placed in 30 DEG C culture 7 days, respectively statistics control pathogen radius and treatment pathogen radius, calculate bacteriostasis rate.

Bacteriostasis rate (%)=(control pathogen radius mm- treatment pathogens radius mm)/control pathogen radius mm × 100。

Streptomycesalbidoflhaving Actin-1 is carried out into Secondary Culture, and determines the 10th generation bacterium, the 20th generation bacterium and the 30th generation bacterium pair Pathogen of Apple Canker (apple black skin shell bacterium) and Botryosphaeria berengeriana, rod method apple specialized form, Phytophthora cactorum, Cherry alternaric bacteria, verticillium dahliae, cherry spherical cavity bacterium, Rhizoctonia solani Kuhn, Botrytis cinerea, anthrax bacteria, apple chain lattice Other 10 kinds of bacteriostasis rates of disease pathogen such as spore, the results are shown in Table 8, and experimental technique is ibid.

The Streptomycesalbidoflhaving Actin-1 of table 8 difference passaged strains are to 11 kinds of bacteriostasis rates of pathogen

As can be seen that Streptomycesalbidoflhaving Actin-1 not only has very strong rejection ability to Pathogen of Apple Canker, To Botryosphaeria berengeriana, rod method apple specialized form, Phytophthora cactorum, cherry alternaric bacteria, verticillium dahliae, cherry spherical cavity 10 kinds of rejection abilities of fruits and vegetables pathogen such as bacterium, Rhizoctonia solani Kuhn, Botrytis cinerea, anthrax bacteria, apple rod method are also very By force, with broad-spectrum antibacterial.

Through passing on micro- Bai Huanglian bacteria strains Actin-1 in 10 generations, 20 generations, 30 generations to 11 kinds of bacteriostasis rates and first of pathogen In generation, compared to almost not reducing, shows that Streptomycesalbidoflhaving Actin-1 of the present invention will not be with the increase of passage number The inhibitory activity to above-mentioned pathogen is reduced, with genetic stability higher.

Influences of the Streptomycesalbidoflhaving Actin-1 of embodiment 4 to Pathogen of Apple Canker mycelia

Streptomycesalbidoflhaving Actin-1 is tested at two flat boards to Pathogen of Apple Canker face-off in picking embodiment 3 Reason group edge mycelia and blank control group mycelia carry out 40 times of microscopies, and microscopy result is shown in Figure 11 and Figure 10, microscopy result table respectively The bright abnormal branch for the treatment of group mycelia increases, and the variation of mycelia ultimate swelling branch, mycelia top or centre have vesicle to produce, also The phenomenon for having part mycelia distortion to assemble, illustrates that Streptomycesalbidoflhaving Actin-1 can suppress Pathogen of Apple Canker spore The sprouting of son can also make pathogen mycelia make a variation so as to suppress pathogen mycelial growth.

The Streptomycesalbidoflhaving Actin-1 of embodiment 5 utilizations mycelial to Pathogen of Apple Canker and induction producing enzyme Research

(1) the mycelial preparation of Pathogen of Apple Canker：See the step 5 of embodiment 1)

(2) preparation method of crude enzyme liquid is：

Activation Streptomycesalbidoflhaving Actin-1, with the step 3 of embodiment 1) streptomycete activation method, and be inoculated with oese To in the 500mL triangular flasks of fluid nutrient medium A, B, C, D equipped with 100mL, every kind of culture medium sets 3 repetitions to one ring, 30 DEG C, 160rpm shaking table cultures, at the 3rd, 5,7 and 9 days in 20mL nutrient solutions are suctioned out with aseptic straw on super-clean bench, will be a small amount of aseptic de- Fat cotton is placed in funnel bottom filtering spore, and filtrate is centrifuged 5min in 4 000rpm, and supernatant is crude enzyme liquid；

The basal liquid medium A quality groups turn into：Pathogen of Apple Canker mycelium：10g, K₂HPO₄· 3H₂O：0.5g, KNO₃：1g, MgSO₄·7H₂O 0.5g, FeSO47H₂O：10mg, distilled water：1L, pH value nature；

The basal liquid medium B quality groups turn into：Tobacco brown spot pathogen：10g, K₂HPO₄·3H₂O：0.5g, KNO₃： 1g, MgSO₄·7H₂O 0.5g, FeSO₄·7H₂O：10mg, distilled water：1L, pH value nature；

The basal liquid medium C quality groups turn into：Starch：10g, K₂HPO₄·3H₂O：0.5g, KNO₃：1g, MgSO₄·7H₂O 0.5g, FeSO₄·7H₂O：10mg, distilled water：1L, pH value nature；

The basal liquid medium D quality groups turn into：Yeast bacterium powder：10g, K₂HPO₄·3H₂O：0.5g, KNO₃：1g, MgSO₄·7H₂O 0.5g, FeSO₄·7H₂O：10mg, distilled water：1L, pH value nature；

Institute it should be noted that：Crude enzyme liquid prepared by basal liquid medium A determines extracellular chitinase (Chitinase), beta-1,3-glucanase (β -1,3-glucosidase), polyphenol oxidase (PPO) and protease (Protease) activity；When crude enzyme liquid prepared by basal liquid medium B is determined using tobacco brown spot pathogen as chitinase inducement Produced chitinase activity；Crude enzyme liquid prepared by basal liquid medium C is determined using starch as beta-1,3-glucanase The beta-1,3-glucanase and polyphenol oxidase activity produced during with polyphenol oxidase inducer；It is prepared by basal liquid medium D Crude enzyme liquid using yeast bacterium powder as protease inducer when the proteinase activity that produces；

As a result：It is micro- when adding Pathogen of Apple Canker mycelium as the sole carbon energy in basal liquid medium A Chitinase activity can be detected in white yellow streptomycete Actin-1 nutrient solutions, enzyme activity is higher.Different incubation time Streptomycesalbidoflhavings The chitinase activity of Actin-1 crude enzyme liquids is different, increases with incubation time, and chitinase activity increases, and the 5th day, enzyme activity reached Peak value, is 140.26U/mL, up to enzymatic activity peak value after with incubation time increase enzymatic activity reduction；In basal liquid medium B Enzyme activity is also the 5th day peaking during with tobacco brown spot pathogen as induction, is 120.47U/mL, less than the mycelial crude enzyme liquid of pathogen Chitinase activity.

As a result：It is micro- when adding Pathogen of Apple Canker mycelium as the sole carbon energy in basal liquid medium A β -1 can be detected in white yellow streptomycete Actin-1 nutrient solutions, 3- dextranase activities, enzyme activity is higher.Enzymatic activity is with incubation time Increase and increase, enzyme activity reached peak value 49.46U/mL at the 7th day, reach as incubation time increases after peak value, enzyme activity drops It is low；It is slightly higher when enzyme activity peakedness ratio is with mycelium as carbon source when in basal liquid medium C with starch as inducer, reach 50.25U/mL。

As a result：It is micro- when adding Pathogen of Apple Canker mycelium as the sole carbon energy in basal liquid medium A Polyphenol oxidase activity can be detected in white yellow streptomycete Actin-1 crude enzyme liquids.Enzymatic activity increasing with incubation time and increasing, and the 7th It when enzymatic activity peaking 39.25U/mL, after peaking with incubation time increase enzyme activity reduction.When in basal liquid medium C During with starch as inducer, the 5th day active peaking of polyphenol oxidase, is 24.91U/mL, is sole carbon source less than mycelium When polyphenol oxidase activity.

As a result：It is micro- white when adding Pathogen of Apple Canker mycelium as the sole carbon nitrogen energy in basal medium A Proteinase activity can be detected in yellow streptomycete Actin-1 nutrient solutions.Enzymatic activity when cultivating the 7th day is 10.90U/mL, shows apple Albumen enzymatic synthesis of the fruit tree putrefaction disease cause of disease mycelium to Streptomycesalbidoflhaving Actin-1 has inducing action.Streptomycesalbidoflhaving Actin-1, using dusty yeast as the 7th day peaking of proteinase activity during the carbon nitrogen energy, is 9.90Um/L in basal medium D, It is little with the protease activity sex differernce produced with the induction of pathogen mycelium.

Table 9 Streptomycesalbidoflhaving Actin-1 streptomycetes, 4 kinds of hydrolase peak activities (U/mL)

(3) Streptomycesalbidoflhaving Actin-1 crude enzyme liquids in ware to the dissolution of pathogen aerial hyphae：

Pathogen of Apple Canker spore suspension is prepared, preparation method is with the step 1 of embodiment 1), the apple that will be prepared Tree rot pathogen spore suspension is spread evenly across on PDA flat boards, is cultivated 8 days at 28 DEG C, treats that plating medium surface is uniform After covering with aerial hyphae, the 3rd prepared by the middle basal liquid medium A of 50 μ L steps (2) is drawn respectively with micropipettor My god, 5 days, the crude enzyme liquid of the Streptomycesalbidoflhaving Actin-1 of 7 days and 9 days, be added dropwise in aerial hyphae table in plate with drop wise fashion Face, while being control with the Proteinase K and sterilized water of 10mg/L, 30 DEG C of culture 72h, observation pathogen aerial hyphae dissolving is existing As.

As a result：Streptomycesalbidoflhaving Actin-1 has obvious dissolution to Pathogen of Apple Canker mycelia, with nothing Bacterium water compares, and is observed during 72h, and the pathogen aerial mycelium of crude enzyme liquid treatment is sunk, and bacteriolyze loop diameter is produced bright up to 6mm Aobvious dissolution.Proteinase K dissolution is most notable, and dissolving loop diameter is 10mm.And sterilized water control CK mycelia do not occur molten Solution phenomenon, only shows the impression of water droplet effect.Illustrate Streptomycesalbidoflhaving Actin-1 in Pathogen of Apple Canker mycelia The lower synthesis hydrolase of body induction and protease have been involved in dissolving the effect of pathogen.

(4) influence of the Streptomycesalbidoflhaving Actin-1 crude enzyme liquids to Pathogen of Apple Canker spore germination

The preparation of Pathogen of Apple Canker spore suspension：Preparation method is with the step 1 of embodiment 1)；

Spore germination is tested：Take in step (2) and cultivate the Streptomycesalbidoflhaving prepared by 7 days with basal liquid medium A The crude enzyme liquid 2mL of Actin-1 is according to 1:The ratio of 4 (V/V) mixes with sterilized water, while the 100 above-mentioned spore suspensions of μ L are accessed, with Sterilized water is control, after 30 DEG C of lucifuge culture 18h, takes 50 μ L and sprouts suspension in 40 times of Microscopic observation spore germination situations, and press Formula calculates germination rate and inhibiting rate；

Germination rate (%)=sprouting spore count/total spore count × 100

Inhibiting rate (%)=(control germination rate-treatment germination rate)/control germination rate × 100

As a result：Basal liquid medium A cultivates the crude enzyme liquid of the Streptomycesalbidoflhaving Actin-1 prepared by 7 days to apple The spore germination for setting rot pathogen has inhibitory action.Sterilized water control treatment, Pathogen of Apple Canker spore germination Rate is 98.60%, and adds the Pathogen of Apple Canker spore germination rate of Streptomycesalbidoflhaving Actin-1 crude enzyme liquids treatment It is only 3.60%, relatively control inhibiting rate improves 96.35%；

(5) Pathogen of Apple Canker mycelium addition produces Substance to Streptomycesalbidoflhaving Actin-1 Influence

By the Pathogen of Apple Canker mycelia scale of construction in basal liquid medium A be respectively set to 5g/L, 10g/L, Tri- concentration treatment of 20g/L, Streptomycesalbidoflhaving Actin-1 is inoculated into equipped with the training of 100mL above threes concentration liquids respectively In foster 500mL triangular flasks, 30 DEG C, 160rpm shaking table cultures 7 days, zymotic fluid are filtered with aseptic 0.22 μm of membrane filter and obtained Bacteria-free filtrate.Bacteria-free filtrate is mixed according to 10% (V/V) ratio with the PDA culture medium for being cooled to 50 DEG C or so, with 10% (V/V) sterilized water is control, is made flat board, in flat board center inoculation 6mm Pathogen of Apple Canker bacteria cakes, every kind of place 3 repetitions of reason, after 7 days, calculate bacteriostasis rate.

Bacteriostasis rate %=(control colony diameter-treatment colony diameter)/(control colony diameter -6mm)

As a result：As shown in table 10 difference mycelium consumption when bacteriostasis rate culture the 7th day when highest.Respectively 52.1%, 83.4% and 96.7%.Improved as the mycelia scale of construction increases bacteriostasis rate.Illustrate that mycelium can induce Streptomycesalbidoflhaving The generation of Actin-1 antibacterial substances.

Different mycelium addition sterile liquid bacteriostasis rate (%) of table 10

Conclusion：During with Pathogen of Apple Canker mycelium as sole carbon source, Streptomycesalbidoflhaving Actin-1 is can induce The fungal cell wall lyase such as synchronized compound chitinase, beta-1,3-glucanase, polyphenol oxidase and protease；Carry significantly Prevention effects of the Streptomycesalbidoflhaving Actin-1 to apple tree canker high；Streptomycesalbidoflhaving Actin-1 crude enzyme liquids are to apple Fruit tree putrefaction disease pathogen mycelia has dissolution.Streptomycesalbidoflhaving during with Pathogen of Apple Canker mycelium as carbon source Actin-1 can produce the stronger antibacterial substance of activity, and bacteriostasis rate is up to 96.7%.

Understand, when apple tree canker affected area applies Streptomycesalbidoflhaving Actin-1 biocontrol agents, when apple tree is rotten Rotten sick pathogen mycelium is contacted with Streptomycesalbidoflhaving Actin-1, and Streptomycesalbidoflhaving Actin-1 can be that carbon source is given birth to it It is long, and the various extracellular cytohydrolists of synthesis are induced, it is pathogen cell disruption by the molten effect of coordinated enzyme, while producing anti- Bacterium active material produces inhibitory action, two kinds of mechanism collective effect tissue apple tree canker diseases to apple rot pathogen growth The breeding of opportunistic pathogen, improves biocontrol effects of the Streptomycesalbidoflhaving Actin-1 to apple tree canker.

Apple tree isolated shoot biological and ecological methods to prevent plant disease, pests, and erosion experiment in the laboratory of embodiment 6

(1) isolated shoot experimental technique：

The preparation method of Streptomycesalbidoflhaving Actin-1 culturing filtrates：

It is prepared by culturing filtrate：5% (V/V) Streptomycesalbidoflhaving Actin-1 spore suspensions of inoculation, the same embodiment of preparation method 2, in the 250mL triangular flasks for filling 50mL Gause I fluid nutrient mediums, 30 DEG C, 180rpm shaking tables concussion and cultivate 8 days.Fermentation After end, first by zymotic fluid 4000rpm be centrifuged after, high fermentation clear liquid through qualitative filter paper coarse filtration, then be equipped with a diameter of 0.45 μ The sand core filter suction filtration of m filter membranes, is finally filtered with the aseptic bacterial filter equipped with a diameter of 0.22 μm of filter membrane again, that is, trained Support filtrate, is fitted into sterilize triangular flask in it is standby in 4 DEG C of Storage in refrigerator.

Prevention effect-isolated shoot the reality of Streptomycesalbidoflhaving Actin-1 bacteria cakes and culturing filtrate to apple tree canker Test；

The collection raw Fuji apple branches of experimental plot 1-2, the branch section that clip 40cm is long, thickness is consistent, are entered with 75% ethanol Row surface sterilization, is then rinsed well with aqua sterilisa, the absorbent cotton moisturizing of two ends moistening.Burn red with the card punch of diameter 6mm After punch, 2 holes of each branch, as vaccination：

1 (preventive effect) of experiment：Streptomycesalbidoflhaving Actin-1 bacteria cakes are inoculated with to above-mentioned vaccination, to be inoculated with sky White culture medium PDA is negative control, removes bacteria cake two days later and is inoculated with the apple rot pathogen bacteria cake of vigorous growth, the treatment It is set to treatment 1；Culturing filtrate is smeared to above-mentioned vaccination, to smear sterilized water as negative control, the phenylate of 0.5g/L is smeared Methyl cyclic-azole solution as positive control (to 100 μ L are first smeared in inoculation hole, room temperature is dried, then smears 100 μ L, and room temperature is dried), The apple rot pathogen bacteria cake of same day inoculation vigorous growth after drying, the treatment is set to treatment 2；2 vaccinations of each branch, Often treatment sets 3 repetitions, 6 vaccinations of each treatment group, above-mentioned vaccination preservative film moisturizing 72h, in 25 DEG C of constant temperature trainings Cultivated 3-14 days in foster case, a situation arises for observation apple tree canker, measures Lesion size, calculate lesion area and preventing and treating and imitate Really, 11 are shown in Table.

2 (governance roles) of experiment：The apple rot pathogen bacteria cake of vigorous growth is inoculated with to above-mentioned vaccination, preservative film is protected After wet 2 days, remove rotten pathogenic bacteria bacteria cake, Streptomycesalbidoflhaving Actin-1 bacteria cakes are then inoculated with, to be inoculated with blank cultures PDA It is negative control, the treatment is set to treatment 1；The apple rot pathogen bacteria cake of vigorous growth is inoculated with to above-mentioned vaccination, it is fresh-keeping After film moisturizing 2 days, remove rotten pathogenic bacteria bacteria cake, smear culturing filtrate, to smear sterilized water as negative control, smear 0.5g/L Difenoconazole solution used as positive control, (to 100 μ L are first smeared in inoculation hole, room temperature is dried, then smears 100 μ L, room temperature Dry) 2 vaccinations of each branch, 3 repetitions of often treatment setting, each 6 vaccination for the treatment of,.Above-mentioned vaccination is with fresh-keeping Film moisturizing 72h, cultivates 3-14 days in 25 DEG C of constant incubators, and a situation arises for observation apple tree canker, and measurement scab is big It is small, calculate lesion area and prevention effect.It is shown in Table 12.

Lesion area=1/4 × Π × major diameter × minor axis

Prevention effect (%)=[(control lesion area-bacteria cake area)-(treatment lesion area-bacteria cake area)]/ (control lesion area-bacteria cake area) × 100

Protective effects of the Streptomycesalbidoflhaving Actin-1 of table 11. to isolated shoot apple tree canker

Note：Difference reaches 5% level of signifiance between different lowercase letters treatment are marked after same column data；

Therapeutic actions of the Streptomycesalbidoflhaving Actin-1 of table 12. to isolated shoot apple tree canker

As can be seen that preventive effect bacteria cake and culturing filtrate preventive effect difference are less, up to more than 95%, bacteria cake is prevented It is 95.50% to imitate, and culturing filtrate is 95.12%.Both therapeutic effects are close, and bacteria cake is treated to apple tree canker isolated shoot Effect is up to 86.05%, and culturing filtrate therapeutic effect is suitable with chemical agent Difenoconazole effect up to 83.48%.

The preparation of the micro- white yellow strepto- Actin-1 biocontrol agents of embodiment 7

1) actication of culture：By Streptomycesalbidoflhaving Actin-1 strains streak inoculation in Gause I agar medium inclined-plane On, 30 DEG C of cultures extremely produce spore in 7 days；

2) prepared by spore suspension：5mL sterilized waters addition length is had on the inclined-plane of Streptomycesalbidoflhaving Actin-1, with sterilizing Transfer needle scrapes spore, adds in sterilized water, and concussion is mixed, and adjustment spore concentration is 1 × 10⁶Individual/mL obtains final product spore suspension；

3) prepared by fermentation seed liquid：Above-mentioned spore suspension is inoculated into liquid seed culture medium by inoculum concentration 1% (v/v) In, 30 DEG C, 200r/min, Shaking culture 48h finally give fermentation seed liquid；

4) solid fermentation：

1. spice:According to solid fermentation culture medium prescription, first with part water by K₂HPO₄、MgSO₄·7H₂O dissolves, then Mixed with other raw materials, packing to sterilizing bag, sterilize 45min in 121 DEG C, is cooled to 35 DEG C；

2. sabot, inoculation, fermentation:By 70cm long, after 121 DEG C of sterilizing 45min of 50cm depth 5cm koji trays wide, load step 1. Solid fermentation culture medium after sterilizing is thick to 1-3cm, the inoculum concentration inoculation step 3 by volume mass than 10%) fermentation seed liquid； The newspaper covering of bottom sterilizing, the sack covering of upper strata sterilizing, moves into bent room；In 30 DEG C of temperature, the culture of humidity 65% 24h Sack, newspaper are raised afterwards, turns over rake, then successively cover newspaper, sack, be repeated once per 24h；Continue to train under similarity condition Support and baking room moved to 96h, dried in 35 DEG C, crush, cross 120 mesh sieves obtain final product original spore powder.

The liquid seed culture medium quality composition：Corn flour：10g, beancake powder：10g, starch：10g, K₂HPO₄：0.2g, Running water：1L, pH value 7.2.

The solid fermentation culture medium quality is constituted：Cotton seed hull：160g, wheat bran：280g, corn flour：120g, beancake powder 120g, calcium carbonate：10g, K₂HPO₄：2g, MgSO₄·7H₂O：2g, quick lime：5g, running water：1L, pH value 7.0；

5) auxiliary agent screening：External environment such as temperature, humidity and illumination etc. can influence stability, the speed of action of microbial inoculum And the persistence of effect, therefore a kind of microorganism formulation of stabilization need to consider different auxiliary agent and auxiliary agent ratio to microbial inoculum activity Influence.Therefore the present invention is screened to auxiliary agent, and carries out variance analysis using DPS statistical softwares and the new multipoles of Duncan are poor Method carries out significance test of difference：

Vector selection：By Streptomycesalbidoflhaving Actin-1 original spore powders and 4 kinds of carriers (calcium carbonate, diatomite, activated carbon, Calcium-base bentonite) mixed in equal amounts obtains 4 kinds of microbial inoculums, and 3 repetitions of every kind for the treatment of are set to treatment group, without the original spore powder of carrier It is control group, 4 kinds of microbial inoculums are placed into 30 DEG C of lucifuge constant temperature is preserved 30 days, and each bacterium is determined using Gause I agar plate count method The bacterium work amount of agent；

As a result：As shown in Table 13, after being preserved 30 days at 30 DEG C, the microbial inoculum bacterium of carrier is added to live than the control of no carrier added Group bacterium work amount is high, wherein the bacterium work amount highest with diatomite as carrier, up to 65.68 × 10¹¹Cfu/g, with other carrier microbial inoculum bacterium Amount significant difference living, therefore screening diatomite is the carrier of Streptomycesalbidoflhaving Actin-1 original spore powders；

Influence of the different carriers to microbial inoculum bacterium work amount under 13 30 DEG C of preservation conditions of table

Note：It is newly multiple through Duncan between being processed with different carriers under 30 DEG C of preservation conditions of different lowercase letters after column data Range method inspection difference work property (P<0.05)；

Dispersant is screened：By Streptomycesalbidoflhaving Actin-1 original spore powders and 4 kinds of dispersants (lauryl sodium sulfate, can Soluble starch, sodium carboxymethylcellulose, sodium lignin sulfonate) respectively according to mass ratio 10:1 and 50:1 ratio mixing, obtains 8 Microbial inoculum is planted, 3 repetitions of every kind for the treatment of are set to treatment group, are control group, 30 DEG C of lucifuge perseverances with the original spore powder without dispersant Temperature is preserved 30 days, and the bacterium work amount of each microbial inoculum is determined using Gause I agar plate count method；

As a result：From table 14 and table 15, original spore powder is 10 with dispersant mass ratio:1 and 50:1 preserve when be with Microbial inoculum bacterium work amount highest, respectively 65.24 × 10 when soluble starch is dispersant¹¹Cfu/g and 89.24 × 10¹¹Cfu/g, with It is notable as dispersant bacterium work amount comparing difference to add lauryl sodium sulfate, sodium carboxymethylcellulose, sodium lignin sulfonate, no The control group bacterium work amount for adding dispersant is minimum, it is known that dispersant can strengthen the stability of microbial inoculum bacterium work amount, therefore screen solvable Property starch for Streptomycesalbidoflhaving Actin-1 original spore powders dispersant；

The dispersant of table 14 and original spore powder 10:1 preserves the influence to microbial inoculum bacterium work amount

Note：Check poor through Duncan duncan's new multiple range methods between being processed with different lowercase letter difference dispersants after column data Different work (P<0.05)；

The dispersant of table 15 and original spore powder 50:1 preserves the influence to microbial inoculum bacterium work amount

Uv-protector is screened：By Streptomycesalbidoflhaving Actin-1 original spore powders and 4 kinds of uv-protectors (humic acid, pastes Essence, sodium alginate and methylcellulose) respectively according to mass ratio 50:1 ratio mixing, obtains 4 kinds of microbial inoculums and is set to treatment group, with Original spore powder without uv-protector is control group, and 1h, every kind for the treatment of 3 are irradiated under every kind of microbial inoculum is placed into uviol lamp respectively Individual repetition, the bacterium work amount of each microbial inoculum is determined using Gause I agar plate count method；

As a result：As shown in Table 16, addition humic acid is the microbial inoculum bacterium work amount highest of uv-protector, be 73.18 × 10¹¹Cfu/g, with sodium alginate, dextrin, methylcellulose as uv-protector bacterium work amount significant difference, without ultraviolet guarantor The control group bacterium work amount for protecting agent is minimum, therefore selects humic acid as the ultraviolet protection of Streptomycesalbidoflhaving Actin-1 original spore powders Agent；

Influence of the uv-protector of table 16 to microbial inoculum bacterium work amount

Note：Examined through Duncan duncan's new multiple range methods between being processed with different lowercase letter difference uv-protectors after column data Test difference work property (P<0.05)；

Orthogonal experiment：Carrier, dispersant and the uv-protector that above-mentioned screening is obtained are L₉(3⁴) orthogonal experiment, it is shown in Table 13, the bacterium work amount of each microbial inoculum is determined using Gause I agar plate count method, screen optimum combination.

The orthogonal of table 17

As a result：Orthogonal result statistical analysis is shown in Table 18, carrier, three kinds of factor extreme difference value difference of dispersant and uv-protector For：20.08th, 18.05,16.39, wherein carrier extreme difference value is maximum, therefore carrier is influence Streptomycesalbidoflhaving Actin-1 spores The biggest factor that former powder bacterium lives, dispersant takes second place, and uv-protector influence is minimum.Comprehensive mean analysis simultaneously, can draw optimal Combination A₁B₂C₃, as can be seen from Table 18, A₁B₂C₃Combination Streptomycesalbidoflhaving Actin-1 microbial inoculums bacterium lives maximum, be 69.00 × 10¹¹Cfu/g, that is, finally giving biocontrol agent composition is：57% Streptomycesalbidoflhaving Actin-1 original spore powders, 40% silicon Diatomaceous earth, 1% soluble starch, 2% humic acid.

The orthogonal of table 18 and result of the test

Being designed by single factor experiment carries out 3 kinds of screenings of auxiliary agent such as carrier, dispersant and uv-protector and in this base Carrier, dispersant and the uv-protector for obtaining will be screened on plinth and uses L₉(3⁴) orthogonal design, final optimization pass obtains micro- Bai Huanglian The formula of mould Actin-1 biocontrol agents：57% Streptomycesalbidoflhaving original spore powder, 40% diatomite, 1% soluble starch Humic acid with 2% is compounded, and finally gives the biocontrol agent of Streptomycesalbidoflhaving Actin-1, using Gause I agar Colony counting method carries out bacterium work measurement to biocontrol agent to be determined, and the bacterium work amount for finally giving biocontrol agent is：6.90×10¹²cfu/g。

Preventive and therapeutic effect-the field experiment of biocontrol agent prepared by 8 example of embodiment 7 to apple tree canker

Field experimental design：Totally 4 treatment, i.e. Streptomycesalbidoflhaving Actin-1 biocontrol agents:The pure land (m/m)=1:1、 Streptomycesalbidoflhaving Actin-1 biocontrol agents:The pure land (m/m)=1:5th, Streptomycesalbidoflhaving Actin-1 biocontrol agents:The pure land (m/m)=1:10；Control group is the pure land.Test site is carried out in Baoji, Shaanxi province city Qianyang County Shaanxi Feng Dan belles apple orchard, Examination tree kind is life " Fuji " in 5 years.

(1) by field experiment, protection and therapeutic effect of the checking Streptomycesalbidoflhaving to apple tree canker

Preventive effect：Life in 5 years, growing way close Fuji apple trees are punched with 6mm card punch on major branch and cause fresh Wound 2cm², 4 kinds process Streptomycesalbidoflhaving Actin-1 biocontrol agents suitable quantity of water and into pureed by more than, are attached to wound and go out Place, the thickness of bacterium mud is 1.5cm or so, and moisturizing is wrapped up with preservative film, after 3 days, is inoculated with the apple rot pathogen of vigorous growth Cake, moisturizing is wrapped up with preservative film, after 7 days, removes preservative film.Often process 3 repetitions, often repeatedly 6 tree.Observed after 4 weeks and rotted The incidence of disease, calculates the incidence of disease, lesion area and prevention effect；Result such as table 19.

Therapeutic action：The close Fuji apple trees of 5 years lifes, growing ways, are punched with 6mm card punch on major branch and cause fresh wounds 2cm², the apple rot pathogen cake of vigorous growth is inoculated with, moisturizing is wrapped up with preservative film, after 3 days, remove rot pathogen bacterium Cake, by 4 kinds of Streptomycesalbidoflhaving Actin-1 biocontrol agents suitable quantity of water for processing above and into pureed, is attached to wound source bacterium The thickness of mud is 1.5cm or so, and moisturizing is wrapped up with preservative film, after 7 days, removes preservative film.3 repetitions are often processed, often repeatedly 6 Tree.The incidence of rot is observed after 4 weeks, the incidence of disease, lesion area and therapeutic effect is calculated；Result such as table 20.

The incidence of disease (%)=diseased plant tree/total strain number

Lesion area=1/4 × Π × major diameter × minor axis

Protective effect of the field Streptomycesalbidoflhaving of table 19 to apple tree canker

Therapeutic action of the field Streptomycesalbidoflhaving of table 20 to apple tree canker

Field control effect shows that Streptomycesalbidoflhaving Actin-1 biocontrol agents and the pure land are with ratio 1：1 and 1:5 mixing During effect, only one tree is caught an illness, and preventive effect is notable, when ratio is 1:When 10, diseased plant tree increases by 1, and effect is also preferable.Control Therapeutic effect shows that Streptomycesalbidoflhaving Actin-1 biocontrol agents and the pure land are with ratio 1：1 and 1:During 5 immixture, apple tree hair Sick rate is 16.67%, and prevention effect is respectively up to 87.80% and 87.60%, and treatment fruit is notable, when ratio is 1:When 10, preventing and treating effect It is really lower slightly, but also reach 95.23%, compared in control, effect is significant；Illustrate that Streptomycesalbidoflhaving Actin-1 can effectively press down The intrusion and extension of rotten pathogenic bacteria processed, act on extremely obvious to apple tree canker field control.

Embodiment 9 passes through healing effect of the field experiment Streptomycesalbidoflhaving to apple tree canker scab

The Curettage for treating experiment of field rot tree scab：Scab position and its surrounding 1cm health position are scraped with scraper To bast, then by Streptomycesalbidoflhaving Actin-1 biocontrol agents and the pure land 1:1 is well mixed, and with water and into pureed, is attached to At rot scar, the thickness of bacterium mud is 1.5cm, after wrap black plastic film moisturizing, 3/group, totally 9, while with the pure land and Mud 3/group, totally 9, starts once to be investigated every month as control, and half a year in later stage or 1 year are once investigated.Note Record scab recurrence and measure the width of callus, calculate prevention effect, the results are shown in Table 21.

Prevention effect (%)=(control recurrence rate-treatment recurrence rate)/control recurrence rate × 100

Observe 18 pieces of scabs, all recurrences of 9 pieces of control group, and lesion area after half a year to increase with time lengthening, treatment group Without recurrence scab, preventive effect is up to 100%.The average injured tissue for the treatment of group 6.36cm wide, callus 10.23cm wide, four after 2 years Nian Hou, callus 17.3cm, and without recurrence, see Figure 12；Illustrate Streptomycesalbidoflhaving Actin-1 biocontrol agents to apple tree The therapeutic effect of rot is effectively and lasting；

Callus effect of the Streptomycesalbidoflhaving of table 21 to field apple tree canker scab

The Streptomycesalbidoflhaving Actin-1 colonization abilities of embodiment 10 are detected

Callus edge skin after 9 apple tree half a year of the treatment group of embodiment 9 is stripped, laboratory guarantor is taken back It is wet, it is separately cultured, Streptomyces albidoflavus Actin-1 are as a result separated on 9 plants of trees, illustrate that the bacterium can be with Colonize in alternaria mali roberts, and it is very high to colonize rate, can for a long time play preventive and therapeutic effect.

Concrete operations are as follows：The apple tree canker callus bark fetched is rinsed well with running water, is cut into 0.25cm²Fritter, is placed in and 30s is soaked in 75% alcohol, then with 4% sodium hypochlorite surface sterilization 3.5min after, be put into aqua sterilisa It is middle to rinse 4 times, at least 1min every time, after blotting excessive moisture with sterilizing filter paper, every 4 fritters are ground with sterilizing mill, then With sterilized water dilution spread in Gause I agar medium, each 3 repetition of tissue, 30 DEG C are cultivated 3-10 days.With last The aqua sterilisa of once flushing is coated on Gause I culture medium, checking tissue surface sterilization situation.

As a result：9 apple trees are separated to streptomycete, and morphology and molecule mirror will be carried out to isolated streptomycete It is fixed, and sequence alignment is carried out with Streptomyces albidoflavus Actin-1, the streptomycete for finally giving and Actin- 1 sequence homology is 100%, it is known that, isolated streptomycete is Streptomycesalbidoflhaving Actin-1.

Understand, present invention Streptomycesalbidoflhaving used not only, field preferable to apple tree canker prevention effect in laboratory Between prevention effect and callus effect it is ideal, can be applied to the multiple regional apple garden of rot.

SEQUENCE LISTING

<110>Shaanxi Feng Dan belles bio tech ltd

<120>One plant of Streptomycesalbidoflhaving and its application in terms of apple tree canker preventing and treating

<130> 2017

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1502

<212> DNA

<213>Streptomycesalbidoflhaving（Streptomyces albidoflavus）CGMCC No.13927

<400> 1

gagagtttga tcctggctca ggacgaacgc cggcggcgtg cttaacacat gcaagtcgaa 60

cgatgaaccg ctttcgagcg gggattagtg gcgaacgggt gagtaacacg tgggcaatct 120

gccctgcact ctgggacaag ccctggaaac ggggtctaat accggatatg accgtctgcc 180

gcatggtgga tggtgtaaag ctccggcggt gcaggatgag cccgcggcct atcagcttgt 240

tggtgaggta gtggctcacc aaggcgacga cgggtagccg acctgagagg gcgaccggcc 300

acactgggac tgggacacgg cccagactcc tacgggaggc agcagtgggg aatattgcac 360

aatgggcgaa agcctgatgc agcgacgccg cgtgagggat gacggccttc gggttgtaaa 420

cctctttcag cagggaagaa gcgaaagtga cggtacctgc agaagaagcg ccggctaact 480

acgtgccagc agccgcggta atacgtaggg cgcaagcgtt gtccggaatt attgggcgta 540

aagagctcgt aggcggcttg tcacgtcggt tgtgaaagcc cggggcttaa ccccgggtct 600

gcagtcgata cgggcaggct agagttcggt aggggagatc ggaattcctg gtgtagcggt 660

gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg gccgatactg 720

acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780

taaacggtgg gcactaggtg tgggcaacat tccacgttgt ccgtgccgca gctaacgcat 840

taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggcggagcat gtggcttaat tcgacgcaac gcgaggaacc ttaccaaggc 960

ttgacataca ccggaaacgt ctggagacag gcgccccctt gtggtcggtg tacaggtggt 1020

gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080

ccttgtcccg tgttgccagc aggcccttgt ggtgctgggg actcacggga gaccgccggg 1140

gtcaactcgg aggaaggtgg ggacgacgtc aagtcatcat gccccttatg tcttgggctg 1200

cacccgtgct acaatggccg gtacaatgag ctgcgatacc gtgaggtgga gcgaatctca 1260

aaaagccggt ctcagttcgg attggggcct gcaactcgac cccatgaagt cggagtcgct 1320

agtaatcgca gatcagcatt gctgcggtga atacgttccc gggccttgta cacaccgccc 1380

gtcacgtcac gaaagtcggt aacacccgaa gccggtggcc caaccccttg tgggagggag 1440

ctgtcgaagg tgggactggc gattgggacg aagtcgtaac aaggtagccg tagaagggcg 1500

at 1502

SEQUENCE LISTING

<110>Shaanxi Feng Dan belles bio tech ltd

<130> 2017

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1286

<212> DNA

<213>Streptomycesalbidoflhaving（Streptomyces albidoflavus）CGMCC No.13927

<400> 1

tgaggtcgtg ctgaccgtgc tgcacgcggg cggcaagttc ggcggcggcg gctacgcggt 60

ctccggcggt ctgcacggcg tcggcgtctc cgtggtcaac gcgctctcca cgcgcgtcgc 120

cgtggagatc cgcaccgacg gctatcactg gacgcagagc tacgagcgcg gcgtcccgac 180

cgcgccgctc gcccagggcg aggccaccga ggagaccggc acctcggtca ccttctgggc 240

cgacggctcg atcttcgaga ccaccgacta ctccttcgag acgctctccc ggcgcttcca 300

ggagatggcg ttcctcaaca agggcctcac catcaagctc accgacgagc gcgcctccgc 360

caaggcgacc gccggtgccg actcggacga ggccacggcg ggcgagcccg aggagccgcg 420

caccgtcacg taccactacg aaggcggcat cgtcgacttc gtgacgtacc tcaactcccg 480

caagggcgag gtcattcacc cgacggtgat cgacatcgag gcggaggaca aggaccggct 540

cctctccgtc gaggtcgcca cgcagtggaa cggcgggtac agcgaggggg tctactcctt 600

cgccaacacc atccacaccc acgagggcgg cacccacgag gagggcttcc gcggcgcgct 660

gaccaccctg gtcaacaagt acgcccggga ccgcaagctg ctgcgcgaga aggacgacaa 720

cctcaccggc gacgacatcc gcgagggcct gacggcgatc atctccgtca agctcggcga 780

gccgcagttc gagggccaga ccaagaccaa gctgggcaac accgaggcca agaccttcgt 840

gcagaaggtg gtctacgagc acctggccga ctggttcgac cggaacccga acgaggccgc 900

ggacatcatc cgcaagggca tcgccgccgc caccgcccgc gtggcggccc gcaaggcgcg 960

cgacctgacc cgccgcaagg gtctgctgga gaccgcctcg ctccccggca agctctcgga 1020

ctgccagtcg aacgacccgg tcaagtgcga gatcttcatc gtcgagggcg actcggccgg 1080

cggctccgcc aagtccggcc gtgacccgat gtaccaggcc atcctgccga tccgcggcaa 1140

gatcctgaac gtcgagaagg cccgcatcga caagatcctg cacaaccagg agatccaggc 1200

gctcatctcg gccttcggca ccggggtcca cgaggacttc gacatcgaga agctccgcta 1260

ccacaagatc atcctgatgg cggacg 1286

Claims

1. one plant of Streptomycesalbidoflhaving (Streptomyces albidoflavus) Actin-1, deposit number is：CGMCC NO.13927。

2. applications of the Streptomycesalbidoflhaving Actin-1 as claimed in claim 1 in terms of apple tree canker preventing and treating.