CN107502584B - One fluorescent pseudomonads and its application in prevention and treatment walnut black spot - Google Patents

One fluorescent pseudomonads and its application in prevention and treatment walnut black spot Download PDF

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CN107502584B
CN107502584B CN201710958437.XA CN201710958437A CN107502584B CN 107502584 B CN107502584 B CN 107502584B CN 201710958437 A CN201710958437 A CN 201710958437A CN 107502584 B CN107502584 B CN 107502584B
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pseudomonas fluorescens
walnut
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black spot
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段春华
李宗泰
牛赡光
张淑静
刘慇
王清海
曲永赟
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Shandong Academy of Forestry
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Abstract

The invention discloses a fluorescent pseudomonads and its applications in prevention and treatment walnut black spot.The bacterial strain is Pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain GH2-1, and deposit number is CGMCC No.14743.Experiment shows that pseudomonas fluorescens strain of the invention has that the speed of growth is fast, resistance is strong, walnut surface can rapid, high volume colonize the features such as, therefore have a good application prospect.The biological control agent prepared by the Pseudomonas fluorescens can not only efficiently prevent and treat walnut blackspot disease, moreover it is possible to effectively facilitate walnut growth, be a kind of biological control agent of great application prospect.

Description

One fluorescent pseudomonads and its application in prevention and treatment walnut black spot
Technical field
The invention belongs to technical field of plant disease biological control.Specifically, the present invention relates to one plant of Pseudomonas Bacterium and its application in prevention and treatment walnut black spot, and the biological control agent using Pseudomonas fluorescens preparation.
Background technique
Walnut black spot [Xanthomonas jugandis (Pierce) Dowson] also known as walnut black rot, in America and Europe Some country's disease incidence are higher.In China, which is distributed widely in Hebei, Shandong, Shanxi, Liaoning, Henan, Jiangsu, Zhejiang, four The province walnut producing region such as river, Yunnan, Shanxi, Gansu.Walnut black spot is by walnut ecliptic pole hair bacillus [Xanthomonas jugandis (Pierce) Dowson] cause, main harm blade, spray, young fruit and floral organ, annual April~August morbidity.After blade is susceptible, Occur the round and small foxiness of polygonal on blade, piece is connected to when serious, water stain shape haloing is with outside scab；Scab on branch Elongated, dark brown is slightly concave to fall into, and keeps upper section withered because Spot expansion surrounds branch when serious；After inflorescence is invaded, dark brown is generated Water stain shape scab；When young fruit is aggrieved, black fleck occurs for fruit face, and no limbus is gradually expanded blackening in blocks, and gos deep into fruit Meat makes entire fruit rot to fall off together with kernel whole blackening.In recent years due to the increase of China's walnut cultivated area, walnut is black Pinta disease problem is also outstanding day by day, and even more serious in the florescence rainfall time on the high side, which causes shedding serious, falls when serious Fruit rate is up to 60% or more, or even has no harvest.
Walnut black spot is prevented and treated at present mainly based on chemical pesticide, but the high residue of chemical pesticide is tight to the pollution of environment Weight.A large amount of pesticides evaporate into air, flow into water body, settling aggregation in the soil, pollute farming animals fishing fruit product, and pass through food The enrichment transfer of object chain generates harm, thus having of still cannot going of chemical prevention and control method at present into the human body, to human body Effect ground solves the problems, such as walnut bacterial black rot.Biological control is that the generation of disease is reduced using the antagonism between species, To safety of human and livestock, environment compatibility is good and pathogen is not likely to produce drug resistance, thus the advantage not having with chemical bactericide, Just gradually becoming the best mode instead of chemical bactericide.And obtain the basis that efficient Antagonistic Fungi is biological control.
Pseudomonas fluorescens (Pseudomonas fluorescens) belongs to Gram-negative bacillus, is common in plant It is as studying a kind of more bacterium in plant rhizosphere growth-promoting bacterium, since its reproduction speed is fast, adapts in rhizosphere and soil Ability is strong, be easy for workers to culture, preparation stabilization, application is convenient, free from environmental pollution, prevents and treats the advantages that plurality of plant diseases, China The Ministry of Agriculture has been classified as one of microbial pesticide and fertilizer variety of registrable registration, is promoted the use of in China.By Yu Qineng generates one or more antibiotic, so to plant disease, especially soil-borne disease such as samping off, root rot, withered Disease etc. has good preventive and therapeutic effect, is a kind of important beneficial microbe resource, has in agricultural production very important Meaning.
Summary of the invention
The object of the present invention is to provide one plant for efficiently preventing and treating the new microorganism of walnut black spot --- fluorescence is false single Born of the same parents' bacteria strain GH2-1.Pseudomonas fluorescens strain of the invention is fast with the speed of growth, resistance is strong, can on walnut surface The features such as rapid, high volume colonizes, therefore have a good application prospect.
Above-mentioned purpose of the invention is achieved through the following technical solutions: bacterial strain provided by the present invention is that fluorescence is false single Born of the same parents bacterium (Pseudomonas fluorescens) bacterial strain GH2-1 is to separate to obtain from the lake mud of Qingdao of Shandong province Laixi City Laixi , China Committee for Culture Collection of Microorganisms's common micro-organisms center has been preserved on September 26th, 2017 (referred to as CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.14743. It is with following biological characteristics: the micro- protuberance of bacterium colony on beef extract culture medium, faint yellow, edge is extended to the outside in undulations Shape, surface is relatively wet, and easy-to-use transfer needle is provoked, and generates water-soluble yellow green pigment, visible yellow green under the irradiation of ultraviolet light skew ray Fluorescence；Microscopy, somatic cells are in the shape of a rod, have polar flagella, can move；Being negative through Gram's staining (is pair with Escherichia coli According to)；Do not produce gemma and pod membrane.
The cultural method or propagation method of Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 of the present invention include:
(1) Nostoc commune Vanch, which saves, uses beef extract culture medium, formula are as follows: beef extract 3g, glucose 2.5g, sodium chloride 5g, 15~20g of agar, distilled water 1000mL, pH are adjusted to 7.4~7.6；
(2) laboratory fluids culture uses beef extract fluid nutrient medium, formula are as follows: beef extract 3g, glucose 2.5g, chlorine Change sodium 5g, distilled water 1000mL, pH are adjusted to 7.4~7.6；
(3) solid culture based formulas: Gu the mass ratio of material and inorganic salts, the solid material and inorganic salts is 98.8: 1.2；Institute It states solid material to be calculated as in mass ratio, rice husk: corn flour: bean cake powder: wheat bran=55: 25: 17: 2；The inorganic salts are by mass percentage It is calculated as, 20% potassium dihydrogen phosphate, 5% magnesium sulfate, 10% ammonium sulfate, 65% precipitated calcium carbonate.
(4) bulk fermentation culture formula: with solid culture based formulas in (3)
The present invention also provides a kind of for preventing and treating the biological control agent of walnut black spot, and the biological control agent includes Pseudomonas fluorescens (P.fluorescens) the bacterial strain GH2-1.
Also, the present invention also provides a kind of for preventing and treating the biological control method of walnut black spot, the biological control Method includes applying above-mentioned Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 to the walnut plant with black spot disease Or above-mentioned biological control agent.
Present invention provides above-mentioned pseudomonas fluorescens strains or above-mentioned biological control agent in prevention and treatment walnut blackspot Purposes in disease.
The preparation method of biological control agent of the present invention the following steps are included:
(1) seed liquor of the pseudomonas fluorescens strain GH2-1 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 25~27 DEG C；
(3) sterile water is added in the culture of step (2) culture to mix, filtrate is seeded to bulk fermentation culture by filtering Base, 25~27 DEG C of room temperature, 85% or more relative humidity fermenting cellar in carry out fermented and cultured.
In one embodiment of the invention, the preparation method comprises the following steps:
(1) the pseudomonas fluorescens strain GH2-1 is transplanted to beef extract fluid nutrient medium, 25~27 DEG C of shaking table oscillations 3~5d of culture obtains seed liquor；
(2) ratio of the seed liquor in mass ratio 10% of step (1) preparation is inoculated into solid medium, 25~27 DEG C 3~5d of lower shaken cultivation；
(3) ratio that sterile water in mass ratio 1: 15 is added in the culture of step (2) culture is mixed, filtering, by filtrate 1: 6 ratio is seeded to bulk fermentation culture medium by volume, 25~27 DEG C of room temperature, 85% or more relative humidity fermenting cellar in 5~6d of fermented and cultured.
Wherein, the beef extract fluid nutrient medium in step (1) is formulated as beef extract 3g, glucose 2.5g, sodium chloride 5g, distilled water 1000mL, pH are adjusted to 7.4~7.6.
Solid medium in step (2) is made of solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.8:1.2；The solid material is calculated as in mass ratio, rice husk: corn flour: bean cake powder: wheat bran=55: 25: 17: 2；The inorganic salts It is by mass percentage 20% potassium dihydrogen phosphate, 5% magnesium sulfate, 10% ammonium sulfate, 65% precipitated calcium carbonate.
The solid medium of the same step of bulk fermentation culture medium (2) in step (3).
Experiment shows that pseudomonas fluorescens strain of the invention is fast with the speed of growth, resistance is strong, in walnut surface energy The features such as enough rapid, high volumes colonize, therefore have a good application prospect.The biological control system prepared by the Pseudomonas fluorescens Agent can not only efficiently prevent and treat walnut blackspot disease, moreover it is possible to effectively facilitate walnut growth, be a kind of life of great application prospect Object prevents and treats preparation.The microorganism formulation can be used as biological pesticide or bio-feritlizer, prevent and treat walnut black spot.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But these embodiments are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into Row modifications or substitutions, but these modifications and replacement are within the scope of the invention.
Embodiment 1
1, the isolation and purification of Pseudomonas fluorescens (P.fluorescens) GH2-1
Pseudomonas fluorescens (P.fluorescens) GH2-1 of the invention is in May, 2013 from Qingdao of Shandong province Lay It is obtained using dilution-plate method and plate streak in separation in the lake mud of the Laixi Xi Shi lake, separation method are as follows:
(1) separation of Pseudomonas fluorescens
The acquisition of mud sample acquires Laixi lake lake mud in Shandong Province Laixi City, mixes, takes back interior and be temporarily stored into 4 DEG C of incubators. Claim 10g mud sample in 90mL sterile water, is placed in 150rpm in 28 DEG C of shaking tables and shakes 10min, be subsequently placed in 30 DEG C of water-baths and incubate 30min is educated, 100 μ L 10 are taken-2、10-3、10-4Dilution is coated on beef extract culture medium flat plate, and each gradient is coated with three and puts down Row is fallen on beef extract culture medium flat plate in the microbial bacteria that 26 DEG C are cultivated the different shape after 2d on picking beef extract culture medium It crosses, bacterium colony growing state is observed in timing.Then plate streak is used, Pseudomonas fluorescence is purified, numbers respectively It saves.
(2) screening of the efficient antagonism Pseudomonas fluorescens of walnut black spot
1. primary dcreening operation: using opposite culture method, PDA (Potato Dextrose Agar) plate is prepared, with punch glimmering The bacteria cake for taking diameter to be 5mm is beaten at light pseudomonad, walnut blackspot pathogen edge, is implanted in the opposite two sides of plate respectively Centre, 28 DEG C of constant temperature incubations observe Pseudomonas fluorescens to the inhibiting effect of pathogen day by day.
2. secondary screening: the pseudomonas fluorescens strain with efficient antagonistic activity screened being carried out secondary screening, is mainly passed through Temperature tolerance, resistance to acid and alkali, drug-resistant test are crossed, the preferable pseudomonas fluorescens strain of patience is screened, carries out potting prevention and treatment examination It tests and field trial.
The present inventor obtains one plant of Pseudomonas fluorescens that can efficiently prevent and treat walnut black spot by a large amount of screening operations (P.fluorescens)GH2-1.It is demonstrated experimentally that the Pseudomonas fluorescens original powder is shown very in prevention and treatment walnut black spot Efficient control efficiency, so that walnut grows fine.Thus, Pseudomonas fluorescens of the invention has wide application prospect Pseudomonas fluorescens new strains can be used for preparing the biological control agent of prevention and treatment walnut black spot disease.
2, bacterial strain is identified
(1) Microbiological Characteristics: the micro- protuberance of bacterium colony on beef extract culture medium, faint yellow, edge is outside in undulations Shape is extended, surface is relatively wet, and easy-to-use transfer needle is provoked, and generates water-soluble yellow green pigment, visible Huang under the irradiation of ultraviolet light skew ray Green fluorescence；Microscopy, somatic cells are in the shape of a rod, have polar flagella, can move；It is negative through Gram's staining (with Escherichia coli For control)；Do not produce gemma and pod membrane.
(2) molecular biological characteristic
The 16s rRNA gene sequencing of Pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain GH2-1 As a result as follows:
TAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAAC GTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCG GACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGT GTAGCCCAGGCCGTAAGGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCT TAGAGTGCCCACCATTACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCAC GACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCAT TGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAG AGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCC ACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCA TTTCACCGCTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTTGTCAGTTTTGAATGCAGTTCCCAGGTT GAGCCCGGGGATTTCACATCCAACTTAACAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTG CACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAATTGCAGAG TATTAATCTACAACCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCT GGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCA GTGTGACTGATCATCCTCTCCGGCCCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATC CGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGT TCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCCAGGAG CAAGCTCCTT。
(3) cellular morphology and physical and chemical experiment result
The cellular morphology and physical and chemical experiment result of 1 Pseudomonas fluorescens of table (P.fluorescens) GH2-1
Experimental project As a result Experimental project As a result
Gram's staining - Carbohydrate produces acid +
Cell shape It is rod-shaped Glucose +
1 μm of cell dia ﹥ - Xylose +
Flagella staining + L-arabinose +
Motility + Mannitol -
The dyeing of class fat granule - Lactose -
Fluorchrome + Glucose fermentation produces gas +
Catalase + Utilize citrate +
Oxidizing ferment + 50 DEG C of growths +
Anaerobic growth - PH 5.7 is grown +
VP test + 7%NaCl growth +
VP ﹤ pH 6 + Starch Hydrolysis -
VP ﹥ pH 7 - Decompose casein +
Nitrate reduction + Gelatin hydrolysate +
Embodiment 2
1, Pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain GH2-1 fermentation process
Beef extract Liquid Culture based formulas are as follows: beef extract 3g, glucose 2.5g, sodium chloride 5g, distilled water 1000mL, pH It is adjusted to 7.4~7.6.
Gu material is calculated as in mass ratio, rice husk: corn flour: bean cake powder: wheat bran=55: 25: 17: 2；Inorganic salt solution is by matter Measure percentage, 20% potassium dihydrogen phosphate, 5% magnesium sulfate, 10% ammonium sulfate, 65% precipitated calcium carbonate；Solid-to-liquid ratio is 98.8: 1.2 (mass ratio).
The a large amount of solid fermentation process of Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1:
1. strain seed liquor culture
By a small amount of bacterium of pseudomonas fluorescens strain (P.fluorescens) GH2-1 picking from test tube slant, ox is moved to In meat extract fluid nutrient medium, 26 DEG C of shaking table 3~5d of shaken cultivation, this is seed liquor.
2. the culture of solids manufacture strain
Seed liquor is inoculated into solid medium (500mL triangular flask) in 10% ratio, 26 DEG C of constant temperature incubations 3~ 5d, intermediate multiple oscillation.
3. a large amount of solid fermentations
2. the culture sterile water of middle solid culture is pressed into 1: 15 dilution proportion, and is filtered with sterile gauze, is gone out thick Slag, as production bacterium solution, inoculative proportion are inoculated in bulk fermentation culture medium by 1: 6.The raw material of inoculation is placed in (26 DEG C of fermenting cellar With 85% or more relative humidity) in 5~6d of fermented and cultured, obtain fermentation liquid, fermentation liquid active bacteria can reach 30~4,000,000,000/gram (viable count cfu/g).Pseudomonas fluorescens original powder is further made, active bacteria can reach 200~25,000,000,000/gram (cfu/g).
Prevention and treatment for clear Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 fermentation liquid to walnut black spot Effect provides foundation for its popularization and application.We in April, 2016~July the Changqing district of Jinan city town Shuan Quan mountain of swords valley village into Test result, is now reported as follows by row test:
1 material to be tested and method
1.1 reagent agent
By Pseudomonas fluorescens (P.fluorescens) the bacterial strain GH2-1 fermentation of 1 fermentation process of embodiment of the present invention production Liquid, active bacteria be 3,500,000,000/gram；72% agricultural streptomycin sulphate wettable powder (production of bioengineering Co., Ltd, Chengdu Hewlett-Packard, It is commercially available).
1.2 test materials and controlling object
Test material is walnut；Controlling object is walnut black spot [Xanthomonas jugandis (Pierce) Dowson]。
1.3 experimental field situations
Experimental field it is located at the Changqing district of Jinan city town the Shuan Quan Fei County mountain of swords Yu Cun Chinese herbaceous peony hickory nut producing region.In this area, generally For the aggrieved rate of plant walnut black spot 60%~70%, injury rate 10%~30% causes fruit 60% or more when serious Consolidation is black caducous, and kernel percent oil content reduces, and seriously affects the yield and quality of walnut；Seedling leaf is aggrieved heavier than big tree, Sick seedling rate is often as high as 80%.Test soil types be cinnamon soil, the content of organic matter 0.75%, pH value 6.8, it is all test it is small Area's cultivation condition and management measure are consistent, and walnut black spot is in early stage when application.
1.4 experimental designs and arrangement
This test totally 3 processing, respectively 10 times of Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 fermentation liquid, 72% 4200 times of liquid of agricultural streptomycin sulphate wettable powder are not administered clear water and compare, 4 repetitions of each processing, each place 60 plants of reason.On April 11st, 2016, May 16, June 15 and July 10, respectively sprinkling is primary, and spray instrument is workers and peasants -16 Type sprayer.
1.5 pilot surveies and calculation method
1.5.1 meteorological condition
1st application (April 11) same day is cloudy, and 3 grades of wind-force, the highest temperature is 21 DEG C, and the lowest temperature is 9 DEG C, relatively Humidity is 55%.2nd application (May 16), the same day is fine, and 3 grades of wind-force, the highest temperature is 26 DEG C, and the lowest temperature is 13 DEG C, phase It is 60% to humidity；3rd application (June 15) same day is fine, and 3 grades of wind-force, the highest temperature is 32 DEG C, and the lowest temperature is 20 DEG C, Relative humidity is 67%；The 4th application (July 10) same day is cloudy, and 3 grades of wind-force, the highest temperature is 31 DEG C, the lowest temperature 22 DEG C, relative humidity 80%.
1.5.2 drug effect and security survey
Efficacy survey: 14d is investigated after last time is administered.Each processing investigates 50 plants, investigates plant leaf, investigation Disease incidence records disease index and calculates disease index.
Security survey: the safety of 7d and 14d observation walnut after being administered in first time occurs if any phytotoxicity, retouches in detail It states symptom of chemical damage and determines phytotoxicity degree by phytotoxicity grading standard.
Walnut black spot grade scale:
0 grade: blade disease-free spot.
1 grade: lesion area accounts for 10% or less blade area.
2 grades: lesion area accounts for the 10%~20% of blade area.
3 grades: lesion area accounts for the 20%~50% of blade area.
4 grades: lesion area accounts for the 50%~80% of blade area.
5 grades: lesion area accounts for 80% of blade area or more, withered.
1.5.3 drug effect calculation method
Control efficiency is calculated by formula (1), (2):
In formula: CK0--- disease index before blank district is administered；
CK1--- disease index after blank district application；
PT0————Disease index before being administered before chemicals treatment；
PT1————Disease index after being administered after chemicals treatment.
1.5.4 walnut is directly affected
Observing medicament, whether there is or not phytotoxicity to walnut, record the type and extent of phytotoxicity.In addition, also to record to walnut growing way It influences.
Phytotoxicity is recorded with following manner:
If (a) phytotoxicity can be measured or calculate, absolute value representation, such as plant height, setting percentage are used.
(b) in other cases, phytotoxicity degree and frequency can be estimated by following two methods:
1. the phytotoxicity degree of every cell is recorded according to phytotoxicity stage division, with-,+, ++, +++, ++++it indicates.
Phytotoxicity stage division:
-: without phytotoxicity；
+: slight phytotoxicity does not influence plant growth；
++: moderate phytotoxicity, it is resilient, phytomass will not be reduced；
+++: moderate phytotoxicity influences plant normal growth, causes a degree of reduction to phytomass；
The serious phytotoxicity of ++++:, plant growth are obstructed, and phytomass reduces serious.
2. evaluating its phytotoxicity percentage by chemicals treatment district compared with blank district.Meanwhile wanting accurate description plant Symptom of chemical damage (dwarfing, chlorisis, deformity etc.).
2 results
Control efficiency of 2.1 reagent agents to walnut black spot
Three kinds of chemicals treatment pronucleus Peach black spot disease incidences are as shown in table 2, to walnut black spot after three kinds of chemicals treatments Control efficiency it is as shown in table 3.Table 3 the results show that Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 fermentation liquid and 72% agricultural streptomycin sulphate wettable powder all has preferable control efficiency to walnut black spot, wherein the former prevention and treatment effect Fruit reaches 70% or more.From the point of view of the disease index of walnut black spot field trial (table 3), 14d after last time is administered, fluorescence Pseudomonad (P.fluorescens) GH2-1 control efficiency is 76.70%, hence it is evident that it is wettable to be higher than 72% agricultural streptomycin sulphate The control efficiency of property pulvis 55.66%.
2 three kinds of chemicals treatment pronucleus Peach black spot disease incidences of table
To the control efficiency of walnut black spot after 3 three kinds of chemicals treatments of table
2.2 walnut security surveys
Through application 7d and 14d observation, each chemicals treatment district is compared with check plot, and walnut growth is normal, and no phytotoxicity generates, and says Bright P. fluorescens fermentation liquid is for examination concentration to walnut safety.
3 brief summaries
3.1 from the point of view of disease index and control efficiency, Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 (preservation Number CGMCC No.14743) there is preferable control efficiency, last time to be administered walnut black spot after 70% or more 14d, it is bright It is aobvious to be better than comparison medicament, significant difference.
3.2 in terms of walnut growing way, and Pseudomonas fluorescens (P.fluorescens) bacterial strain GH2-1 has walnut apparent Promote growth result, without phytotoxicity, securely and reliably.
SEQUENCE LISTING
<110>Shandong Forest Science Academy
<120>one fluorescent pseudomonads and its application in prevention and treatment walnut black spot
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1370
<212> DNA
<213>the 16s rRNA gene order of Pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain GH2-1
<400> 1
tagactagct acttctggtg caacccactc ccatggtgtg acgggcggtg tgtacaaggc 60
ccgggaacgt attcaccgcg acattctgat tcgcgattac tagcgattcc gacttcacgc 120
agtcgagttg cagactgcga tccggactac gatcggtttt atgggattag ctccacctcg 180
cggcttggca accctttgta ccgaccattg tagcacgtgt gtagcccagg ccgtaagggg 240
ccatgatgac ttgacgtcat ccccaccttc ctccggtttg tcaccggcag tctccttaga 300
gtgcccacca ttacgtgctg gtaactaagg acaagggttg cgctcgttac gggacttaac 360
ccaacatctc acgacacgag ctgacgacag ccatgcagca cctgtctcaa tgttcccgaa 420
ggcaccaatc catctctgga aagttcattg gatgtcaagg cctggtaagg ttcttcgcgt 480
tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcatttgagt 540
tttaaccttg cggccgtact ccccaggcgg tcaacttaat gcgttagctg cgccactaag 600
agctcaaggc tcccaacggc tagttgacat cgtttacggc gtggactacc agggtatcta 660
atcctgtttg ctccccacgc tttcgcacct cagtgtcagt atcagtccag gtggtcgcct 720
tcgccactgg tgttccttcc tatatctacg catttcaccg ctacacagga aattccacca 780
ccctctacca tactctagct tgtcagtttt gaatgcagtt cccaggttga gcccggggat 840
ttcacatcca acttaacaaa ccacctacgc gcgctttacg cccagtaatt ccgattaacg 900
cttgcaccct ctgtattacc gcggctgctg gcacagagtt agccggtgct tattctgtcg 960
gtaacgtcaa aattgcagag tattaatcta caacccttcc tcccaactta aagtgcttta 1020
caatccgaag accttcttca cacacgcggc atggctggat caggctttcg cccattgtcc 1080
aatattcccc actgctgcct cccgtaggag tctggaccgt gtctcagttc cagtgtgact 1140
gatcatcctc tccggcccag ttacggatcg tcgccttggt gagccattac ctcaccaact 1200
agctaatccg acctaggctc atctgatagc gcaaggcccg aaggtcccct gctttctccc 1260
gtaggacgta tgcggtatta gcgttccttt cgaaacgttg tcccccacta ccaggcagat 1320
tcctaggcat tactcacccg tccgccgctg aatccaggag caagctcctt 1370

Claims (7)

1. Pseudomonas fluorescens (Pseudomonas fluorescens) bacterial strain GH2-1, the deposit number of the bacterial strain is CGMCC No.14743。
2. application of the pseudomonas fluorescens strain GH2-1 described in claim 1 in prevention and treatment walnut black spot.
3. a kind of biological control agent including pseudomonas fluorescens strain GH2-1 described in claim 1.
4. the preparation method of biological control agent as claimed in claim 3, characterized in that the following steps are included:
(1) seed liquor of pseudomonas fluorescens strain GH2-1 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 25 ~ 27 DEG C；
(3) sterile water being added in the culture of step (2) culture to mix, filtrate is seeded to bulk fermentation culture medium by filtering, 25 ~ 27 DEG C, carry out fermented and cultured in the fermenting cellar of 85% or more relative humidity；
Solid medium in the step (2) is made of solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.8:1.2；The solid material is calculated as in mass ratio: rice husk: corn flour: bean cake powder: wheat bran=55: 25: 17: 2；The inorganic salts are pressed Mass percent is calculated as: potassium dihydrogen phosphate 20%, magnesium sulfate 5%, ammonium sulfate 10%, precipitated calcium carbonate 65%；
The solid medium of the same step of bulk fermentation culture medium (2) in the step (3).
5. the preparation method of biological control agent as claimed in claim 4, characterized in that the following steps are included:
(1) pseudomonas fluorescens strain GH2-1 is transplanted to beef extract fluid nutrient medium, 25 ~ 27 DEG C of 3 ~ 5 d of shaking table shaken cultivation Obtain seed liquor；
(2) ratio of the seed liquor in mass ratio 10% of step (1) preparation is inoculated into solid medium, is vibrated at 25 ~ 27 DEG C Cultivate 3 ~ 5 d；
(3) ratio that sterile water in mass ratio 1: 15 is added in the culture of step (2) culture is mixed, filtrate is pressed body by filtering Ratio of the product than 1: 6 is seeded to bulk fermentation culture medium, 25 ~ 27 DEG C of room temperature, 85% or more relative humidity fermenting cellar in ferment training Support 5 ~ 6 d.
6. application of the biological control agent as claimed in claim 3 in prevention and treatment walnut black spot.
7. a kind of for preventing and treating the biological control method of walnut black spot, the biological control method includes to black spot disease Walnut plant apply pseudomonas fluorescens strain GH2-1 described in claim 1 or biological control as claimed in claim 3 Preparation.
CN201710958437.XA 2017-10-16 2017-10-16 One fluorescent pseudomonads and its application in prevention and treatment walnut black spot Active CN107502584B (en)

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CN114891679B (en) * 2022-05-19 2023-06-20 山东省林业科学研究院 Pseudomonas fluorescens and application thereof in preventing and controlling cherry branch diseases
CN116042468B (en) * 2022-12-28 2024-04-26 云南大学 Multifunctional fluorescent pseudomonas with functions of promoting pasture growth and low-temperature composting fermentation

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