CN108102991A - One plant of Bei Laisi bacillus and its application in watermelon blight is prevented - Google Patents
One plant of Bei Laisi bacillus and its application in watermelon blight is prevented Download PDFInfo
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- CN108102991A CN108102991A CN201810184045.7A CN201810184045A CN108102991A CN 108102991 A CN108102991 A CN 108102991A CN 201810184045 A CN201810184045 A CN 201810184045A CN 108102991 A CN108102991 A CN 108102991A
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- bei laisi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- Pest Control & Pesticides (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses one plant of Bei Laisi bacillus and its applications in watermelon blight is prevented.The bacterial strain of the present invention is Bei Laisi bacillus (Bacillus velezensis) strain X T1 3, it separates and obtains from the Dongping hu lake mud of Shandong Province Tai'an, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved within 26th in September in 2017, preserving number is CGMCC No.14746.Result of the test shows：3 biological control agents of Bei Laisi Bacillus strains XT1 prevent watermelon blight disease significant effect, and control effect reaches 77.03%, and watermelon growing is healthy and strong, and no poisoning generates.
Description
Technical field
The invention belongs to technical field of plant disease biological control.Specifically, the present invention relates to one plant of Bei Laisi gemma
Bacillus and its application in watermelon blight is prevented and the biological control agent using Bei Laisi bacillus preparation.
Background technology
Watermelon occupies an important position always in world's horticulture, and Chinese growth of watermelon area accounts for world's gross area
55%, total output accounts for more than 70%.In recent years, since growth of watermelon area constantly expands, cause crop rotation difficult, cause to the west of
Cucurbit wilt etc. also aggravates year by year for the disease of representative.Watermelon blight is by watermelon specialized form Fusarium oxysporum (Fusarium
Oxysporum f.sp.niveum) caused by crushing soil-borne disease, be distributed in each watermelon producing region in the world.Watermelon blight is
A kind of vascular bundle diseases can occur in entire growth period, using the bloom phase of bearing fruit and fruit expanding period as onset peak.Due to disease
Opportunistic pathogen can be into breeding in people's plant body and incubation period is long, and the disease is difficult to prevent for a long time, especially in watermelon continuous crop
In the case of, disease occurs even more serious.
Prevention for watermelon blight relies primarily on chemical pesticide in production at present and is prevented.High-toxic pesticide makes
With the pathogen is made to generate the serious resistance to the action of a drug, general chemical prevention cannot solve the problems, such as, give soil environment band instead
Carry out harmful effect.Therefore it is necessary to develop new and effective environmentally friendly biological agent.
Bacillus is the general name of a kind of aerobic or amphimicrobian production gemma Gram-positive bacillus, and physiological characteristic is rich
It is rich various, it is widespread in nature, is the important microbe population of soil and plant surface rhizosphere, to the nontoxic nothing of people and animals
Evil, it is free from environmental pollution, a variety of antibiotic and enzyme can be generated, there is broad spectrum antibiotic activity.Bacillus prevents as biocontrol microorganisms to be planted
The mechanism of object disease mainly has：Antibiosis, nutrient competition, competition for space, hyperparasitism and induction plant generate resistance
Etc. mechanism, main Biocontrol Mechanism be with generate antagonistic substance inhibit harmful levels of pathogens growth or kill pathogen.These objects
Most matter is peptide antibiotics, has efficient, low-residual and advantages of environment protection.
The content of the invention
The object of the present invention is to provide a kind of for efficiently preventing new microorganism --- the Bei Laisi buds of watermelon blight
Spore bacillus strain XT1-3.The present invention Bei Laisi Bacillus strains have the speed of growth is fast, sporulation quantity is big, resistance is strong,
Plant (watermelon) surface can rapid, high volume the features such as colonizing, therefore have a good application prospect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：Bacterial strain provided by the present invention is Bei Laisi buds
Spore bacillus (Bacillus velezensis) strain X T1-3, separates from the Dongping hu lake mud of Shandong Province Tai'an and obtains,
In September in 2017 be preserved within 26th China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC,
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preserving number CGMCC
No.14746.It is with following biological characteristics：On beef extract culture medium bacterium colony be creamy white, mucus, corrugated protrusion,
Microscopy, somatic cells are in rod-shaped, can be moved；It is positive (using Escherichia coli as control) through Gram's staining；After spore staining
Observe that gemma is oval or column.
The cultural method or propagation method of Bei Laisi bacillus (B.velezensis) strain X T1-3 of the present invention includes：
(1) Nostoc commune Vanch is preserved using beef extract culture medium, is formulated and is：Beef extract 3g, glucose 2.5g, sodium chloride
5g, agar 17g, distilled water 1000mL, pH are adjusted to 7.2~7.4.
(2) laboratory fluids culture uses beef extract fluid nutrient medium, is formulated and is：Beef extract 3g, glucose 2.5g, chlorine
Change sodium 5g, distilled water 1000mL, pH are adjusted to 7.2~7.4.
(3) solid culture based formulas：Gu the mass ratio of material and inorganic salts, the solid material and inorganic salts is 98.5: 0.5；Institute
It states solid material and is calculated as corn flour in mass ratio: bean cake powder: wheat bran=65: 25: 10；The component and weight ratio of the inorganic salts be：Phosphorus
Acid dihydride potassium 15%, magnesium sulfate 5%, ammonium sulfate 15%, precipitated calcium carbonate 65%.
(4) bulk fermentation culture formula：With solid culture based formulas in (3).
The present invention also provides a kind of for preventing the biological control agent of watermelon blight, the biological control agent bag
Include Bei Laisi bacillus (B.velezensis) the strain X T1-3.
The present invention also provides a kind of for preventing the biological control method of watermelon blight, the biological control method bag
It includes to the watermelon with droop and applies above-mentioned Bei Laisi bacillus (B.velezensis) strain X T1-3 or above-mentioned biology
Prevent preparation.
Present invention provides above-mentioned Bei Laisi Bacillus strains XT1-3 or above-mentioned biological control agent in prevention west
Purposes in cucurbit wilt.
The preparation method of biological control agent of the present invention, comprises the following steps：
(1) seed liquor of the Bei Laisi Bacillus strains XT1-3 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 27~29 DEG C；
(3) culture of step (2) culture is added in sterile water to mix, filtrate is seeded to bulk fermentation culture by filtering
Base, 27~29 DEG C of room temperature, relative humidity more than 85% fermenting cellar in carry out fermented and cultured.
In one embodiment of the invention, the preparation method comprises the following steps：
(1) spore of the Bei Laisi Bacillus strains XT1-3 is transplanted to beef extract fluid nutrient medium, 27~29 DEG C
3~5d of shaking table shaken cultivation obtains seed liquor；
(2) ratio for the seed liquor in mass ratio 10% for preparing step (1) is inoculated into solid medium, 27~29 DEG C
3~5d of lower shaken cultivation；
(3) culture of step (2) the culture ratio of sterile water in mass ratio 1: 15 is mixed, filtrate is pressed in filtering
The ratio of volume ratio 1: 6 is seeded to bulk fermentation culture medium, 27~29 DEG C of room temperature, relative humidity more than 85% fermenting cellar in send out
8~9d of ferment culture.
Wherein, the beef extract fluid nutrient medium in step (1), is formulated and is：Beef extract 3g, glucose 2.5g, sodium chloride
5g, distilled water 1000mL, pH are adjusted to 7.2~7.4.
Solid medium in step (2) includes expecting admittedly and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.5:
0.5；The solid material is calculated as corn flour in mass ratio: bean cake powder: wheat bran=65: 25: 10；The component and weight ratio of the inorganic salts
For：Potassium dihydrogen phosphate 15%, magnesium sulfate 5%, ammonium sulfate 15%, precipitated calcium carbonate 65%.
The solid medium of the same step of bulk fermentation culture medium (2) in step (3).
Experiment show Bei Laisi Bacillus strains of the invention have the speed of growth is fast, sporulation quantity is big, resistance is strong,
Plant (especially watermelon) surface can rapid, high volume colonize the features such as, therefore have a good application prospect.By the shellfish Lay
Biological control agent prepared by this bacillus can efficiently prevent watermelon blight, be a kind of biology of great application prospect
Prevent preparation.Result of the test shows：Bei Laisi bacillus (B.velezensis) strain X T1-3 biological control agents prevention west
Cucurbit wilt disease significant effect, control effect reaches 77.03%, and watermelon growing is healthy and strong, and no poisoning generates.
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 only exemplary, and do not form any restrictions to the scope of the present invention.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are within the scope of the present invention.
Embodiment 1
1st, the isolation and purification of Bei Laisi bacillus (B.velezensis) XT1-3
Bei Laisi bacillus (B.velezensis) XT1-3 of the present invention is using dilution-plate method peace from lake mud
The separation of plate method of scoring obtains, and separation method is：
(1) separation of bacillus
Lake mud sample is gathered on April 21st, 2013 from Shandong Province Tai'an Dongping hu.Weigh 1g soil samples (lake mud) in
In 100mL sterile waters, it is placed in 150rpm in 30 DEG C of shaking tables and shakes 10min, be subsequently placed in 60 DEG C of water-baths and be incubated 30min, take
100μL 10-2、10-3、10-4Dilution is coated on LB culture medium flat plates, and each gradient coating three is parallel, in 28 DEG C of cultures
The microbial bacteria of different shape after 2d on picking beef extract culture medium falls within and rules on beef extract culture medium flat plate, timing
Observe colony growth situation.Then using plate streak, Bacillus strain is purified, number preserves respectively.
(2) screening of the efficient antagonistic Bacillus of watermelon blight
1. primary dcreening operation：Using opposite culture method, PDA (Potato Dextrose Agar) tablet is prepared, with card punch in bud
Spore bacillus, watermelon blight pathogen edge take the bacteria cake of a diameter of 5mm, be implanted in respectively tablet it is opposite both sides center, 28
DEG C constant temperature incubation observes inhibitory action of the bacillus to pathogen day by day.
2. secondary screening：The Bacillus strain with efficient antagonistic activity screened is subjected to secondary screening, mainly by resistance to
Warm nature, resistance to acid and alkali, drug-resistant test screen the preferable Bacillus strain of patience, carry out potting controlling experiment and field
Experiment, and the strain X T1-3 obtained to screening is identified.
The present inventor obtains one plant of Bei Laisi gemma bar that can efficiently prevent watermelon blight by a large amount of screening operations
Bacterium (B.velezensis) XT1-3.It is demonstrated experimentally that the Bei Laisi fermentation of bacillus liquid is shown in watermelon blight is prevented
Very efficient control effect.Thus, Bei Laisi bacillus of the invention is the Bei Laisi gemma for having wide application prospect
Bacillus new strains can be used for the biological control agent for preparing prevention watermelon blight.
2nd, bacterial strain is identified
(1) Microbiological Characteristics
On beef extract culture medium bacterium colony be creamy white, mucus, corrugated protrusion, microscopy, somatic cells be in rod-shaped, energy
Movement；It is positive (using Escherichia coli as control) through Gram's staining；Observe that gemma is oval or column after spore staining.
(2) molecular biological characteristic
The 16s rDNA gene sequencings result of the bacterial strain is following (SEQ-1)：
CCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGC
GGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAG
ATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCA
TAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTG
AATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAA
CCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTA
AGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCA
GTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACT
TAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTC
AGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATT
CCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGA
CTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGC
TGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCC
TAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGA
AGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCG
GCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGC
CGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCC
AGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCC。
(3) cellular morphology and physical and chemical experiment result
The cellular morphology and physical and chemical experiment result of table 1 Bei Laisi bacillus (B.velezensis) XT1-3
| Experimental project | As a result | Experimental project | As a result |
| Gram's staining | It is positive | Carbohydrate production acid | + |
| Cell shape | It is rod-shaped | Glucose | + |
| 1 μm of cell dia ﹥ | - | Xylose | + |
| Form gemma | + | L-arabinose | + |
| Gemma expands | - | Mannitol | - |
| Gemma is circular | - | Lactose | - |
| Parasporal crystal | - | Glucose fermentation aerogenesis | + |
| Catalase | + | Utilize citrate | + |
| Oxidizing ferment | + | 50 DEG C of growths | + |
| Anaerobic growth | - | PH 5.7 is grown | + |
| VP is tested | + | 7%NaCl is grown | + |
| VP ﹤ pH 6 | + | Starch Hydrolysis | + |
| VP ﹥ pH 7 | - | Decompose casein | + |
| Nitrate reduction | + | Gelatin hydrolysate | + |
Strain X T1-3 is accredited as Bei Laisi bacillus, is preserved in Chinese microorganism strain within 26th in September in 2017
Preservation administration committee common micro-organisms center, preserving number are CGMCC No.14746.
Embodiment 2
1st, fermentation of bacillus process
Beef extract Liquid Culture based formulas is：Beef extract 3g, glucose 2.5g, sodium chloride 5g, distilled water 1000mL, pH
It is adjusted to 7.2~7.4.
Solid culture based formulas：Gu the mass ratio of material, inorganic salts and water, the solid material and inorganic salts is 98.5: 0.5；Institute
It states solid material and is calculated as corn flour in mass ratio: bean cake powder: wheat bran=65: 25: 10；The component and weight ratio of the inorganic salts be：Phosphorus
Acid dihydride potassium 15%, magnesium sulfate 5%, ammonium sulfate 15%, precipitated calcium carbonate 65%.Preparation method：Add in suitable water (10-15
Times quality) dissolving inorganic salts, obtain inorganic salt solution；Inorganic salt solution is mixed with solid material, and adds in the training of water final adjustment solid
The water content of base is supported as 40-60%, solid medium is held in the hand, has water in webs but not drippage is advisable.
The same solid medium of bulk fermentation culture medium.
The a large amount of solid fermentation process of Bei Laisi bacillus (B.velezensis) strain X T1-3：
1. strain seed liquor culture
By a small amount of spore of Bei Laisi Bacillus strains (B.velezensis) XT1-3 pickings from test tube slant, move to
In beef extract fluid nutrient medium, 28 DEG C of shaking table 3~5d of shaken cultivation, this is seed liquor.
2. the culture of solids manufacture strain
Seed liquor is inoculated into 10% ratio in solid medium (500mL triangular flasks), 28 DEG C of constant temperature incubations 3~
5d, intermediate multiple oscillation.
3. a large amount of solid fermentations
It by the culture of 2. middle solid culture with sterile water by 1: 15 dilution proportion, and is filtered, removed thick with sterile gauze
Slag, as produces bacterium solution, and inoculative proportion is inoculated in bulk fermentation culture medium by 1: 6.The raw material of inoculation is placed in (28 DEG C of fermenting cellar
With relative humidity more than 85%) in 8~9d of fermented and cultured, solid fermentation culture active bacteria can reach 1,500,000,000/gram.
For prevention of clear and definite Bei Laisi bacillus (B.velezensis) the strain X T1-3 zymotic fluids to watermelon blight
Effect provides foundation for its popularization and application.We have carried out crop field examination in June, 2016 in Heze city of Shandong province Dongming County
It tests, is now reported as follows result of the test：
1 material to be tested and method
1.1 reagent agent
Bei Laisi bacillus (B.velezensis) the strain X T1-3 produced by 2 fermentation process of the embodiment of the present invention consolidates
Body fermentation culture medium (1,500,000,000/gram)；(Shandong rural area bio tech ltd produces 25% carbendazol wettable powder, city
It sells).
1.2 materials to be tested and controlling object
Material to be tested is watermelon；Controlling object is watermelon blight.
1.3 experimental field situations
Experimental field it is located at Heze city of Shandong province Dongming County Datun Town watermelon producing region.In this area, due to continuous cropping continuous cropping, watermelon
Droop causes very big economic loss to peasant than more serious.Experiment soil types is moisture soil, and the content of organic matter is
0.63%, pH value 7.4, all experimental plot cultivation conditions and management measure are consistent, and watermelon blight is in morbidity during dispenser
Initial stage.
1.4 experimental designs and arrangement
This experiment totally 3 processing, are respectively 20 times of Bei Laisi bacillus (B.velezensis) strain X T1-3 zymotic fluids
Liquid, 25% carbendazol wettable powder, 500 times of liquid, not dispenser clear water compare, and each handle 4 repetitions, each handle 140
Strain.To diseased plant and its neighbouring plant pouring root and full field foliar spray is carried out in early stage, 10 days once, continuous 2 times.It is every every time
Diseased plant irrigation liquid 0.25kg；Full field spraying, per 666.7m2It is 30~50L with liquid volume.On June 1st, 2016 and June 10
Once, spray instrument is the type sprayer of workers and peasants -16 for each pouring root, sprinkling.
1.5 pilot surveies and computational methods
1.5.1 meteorological condition
1st dispenser (June 1) same day is cloudy, 3 grades of wind-force, and the highest temperature is 30 DEG C, and the lowest temperature is 19 DEG C, relatively
Humidity is 67%.2nd dispenser (June 10), the same day is cloudy, 3-4 grades of wind-force, and the highest temperature is 33 DEG C, the lowest temperature 22
DEG C, relative humidity 70%.
1.5.2 drug effect and security survey
Efficacy survey：14d is investigated after last time dispenser.Each 120 plants of processing investigation, investigates plant vines base
Portion, Investigate incidence record disease index and calculate disease index.
Security survey：The security of 7d and 14d observations watermelon, occurs if any poisoning, retouches in detail after first time dispenser
It states symptom of chemical damage and determines poisoning degree by poisoning grading standard.
Stage division (in units of single plant)：
0 grade：Watermelon plant health, disease-free spot.
1 grade：Occur slight scab in watermelon plants stems, but grow normal.
3 grades：Scab is apparent in watermelon plants stems, and growth is slight suppressed.
5 grades：Scab is apparent in watermelon plants stems, and plant top is wilted, slow-growing or rigid.
7 grades：More than 50% browning of watermelon plant is withered.
9 grades：More than 50% browning of watermelon plant is withered or withered.
1.5.3 drug effect computational methods
Drug effect is calculated by formula (1), (2)：
In formula：CK0--- disease index before blank district dispenser；
CK1--- disease index after blank district dispenser；
PT0————Disease index before chemicals treatment dispenser；
PT1————Disease index after chemicals treatment dispenser.
1.5.4 to the influence of watermelon growing
Observation medicament whether there is poisoning to watermelon, records the type and extent of poisoning.In addition, also to record to watermelon other
Beneficial effect (such as stimulating growth etc.).
Poisoning is recorded with following manner：
If (a) poisoning can be measured or calculate, with absolute value representation, such as plant height.
(b) in other cases, poisoning degree and frequency can be estimated by following two methods：
1. the poisoning degree of watermelon is recorded according to poisoning stage division, with-,+, ++, +++, ++++it represents.
Poisoning stage division：
—：Without poisoning；
+：Slight poisoning, does not influence plant growth；
++：Moderate poisoning, it is resilient, crop failure will not be caused；
+++：Moderate poisoning influences crop normal growth, and a degree of loss is caused to crop yield and quality；
++++：Serious poisoning, plant growth are obstructed, and yield and quality loss is serious.
2. by chemicals treatment district compared with blank district, its poisoning percentage is evaluated.Meanwhile want accurate description crop
Symptom of chemical damage (dwarfing, chlorisis, deformity etc.).
2 results
2.1 reagent agents are to the control effect of watermelon blight
Reagent agent is to the control effect of watermelon blight as shown in table 2-3.Table 3 the result shows that, Bei Laisi bacillus
(B.velezensis) strain X T1-3 solid fermentation cultures and 25% carbendazol wettable powder have to watermelon blight compared with
Good control effect, wherein the former control effect reach more than 75%.
From the point of view of the disease index of watermelon blight field trial (table 3), 14d after last time dispenser, Bei Laisi gemma
Bacillus (B.velezensis) XT1-3 control effects are 77.03%, hence it is evident that higher than 25% carbendazol wettable powder 53.63%
Control effect.
Watermelon blight disease incidence before 2 three kinds of chemicals treatments of table
3 three kinds of chemicals treatments of table are to the control effect of watermelon blight
2.2 watermelon security surveys
It is observed through dispenser 7d and 14d, for each chemicals treatment district compared with check plot, watermelon growing is normal, and no poisoning generates, and says
Ming Beilaisi bacillus solid fermentation culture is for examination concentration to watermelon safety.
3 brief summaries
3.1 from the point of view of disease index and control effect, and Bei Laisi bacillus (B.velezensis) strain X T1-3 (is protected
Tibetan CGMCC No.14746) there is preferable control effect, the control effect of 14d after last time dispenser to watermelon blight
Up to more than 75%.
3.2 in terms of watermelon growing way, and Bei Laisi bacillus (B.velezensis) strain X T1-3 has watermelon apparent
Promote growth result, without poisoning, securely and reliably.
SEQUENCE LISTING
<110>Shandong Dai Meng obtains bio tech ltd
<120>One plant of Bei Laisi bacillus and its application in watermelon blight is prevented
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1376
<212> DNA
<213>Bei Laisi bacillus（Bacillus velezensis）The 16s rDNA gene orders of strain X T1-3
<400> 1
cctcaccgac ttcgggtgtt acaaactctc gtggtgtgac gggcggtgtg tacaaggccc 60
gggaacgtat tcaccgcggc atgctgatcc gcgattacta gcgattccag cttcacgcag 120
tcgagttgca gactgcgatc cgaactgaga acagatttgt gggattggct taacctcgcg 180
gtttcgctgc cctttgttct gtccattgta gcacgtgtgt agcccaggtc ataaggggca 240
tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca ccttagagtg 300
cccaactgaa tgctggcaac taagatcaag ggttgcgctc gttgcgggac ttaacccaac 360
atctcacgac acgagctgac gacaaccatg caccacctgt cactctgccc ccgaagggga 420
cgtcctatct ctaggattgt cagaggatgt caagacctgg taaggttctt cgcgttgctt 480
cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcctt tgagtttcag 540
tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt agctgcagca ctaaggggcg 600
gaaaccccct aacacttagc actcatcgtt tacggcgtgg actaccaggg tatctaatcc 660
tgttcgctcc ccacgctttc gctcctcagc gtcagttaca gaccagagag tcgccttcgc 720
cactggtgtt cctccacatc tctacgcatt tcaccgctac acgtggaatt ccactctcct 780
cttctgcact caagttcccc agtttccaat gaccctcccc ggttgagccg ggggctttca 840
catcagactt aagaaaccgc ctgcgagccc tttacgccca ataattccgg acaacgcttg 900
ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtggctttct ggttaggtac 960
cgtcaaggtg ccgccctatt tgaacggcac ttgttcttcc ctaacaacag agctttacga 1020
tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc attgcggaag 1080
attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag tgtggccgat 1140
caccctctca ggtcggctac gcatcgtcgc cttggtgagc cgttacctca ccaactagct 1200
aatgcgccgc gggtccatct gtaagtggta gccgaagcca ccttttatgt ctgaaccatg 1260
cggttcagac aaccatccgg tattagcccc ggtttcccgg agttatccca gtcttacagg 1320
caggttaccc acgtgttact cacccgtccg ccgctaacat cagggagcaa gctccc 1376
Claims (8)
- Bei Laisi bacillus 1. (Bacillus velezensis) strain X T1-3, the deposit number of the bacterial strain is CGMCC No.14746。
- 2. purposes of the Bei Laisi Bacillus strains XT1-3 described in claim 1 in watermelon blight is prevented.
- 3. a kind of biological control agent, including Bei Laisi Bacillus strains XT1-3 described in claim 1.
- 4. the preparation method of biological control agent described in claim 1, it is characterized in that, comprise the following steps：(1) seed liquor of Bei Laisi Bacillus strains XT1-3 is prepared；(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 27~29 DEG C；(3) culture of step (2) culture being added in sterile water to mix, filtrate is seeded to bulk fermentation culture medium by filtering, 27~29 DEG C of room temperature, relative humidity more than 85% fermenting cellar in carry out fermented and cultured.
- 5. the preparation method of biological control agent as claimed in claim 4, it is characterized in that,Solid medium in the step (2) includes expecting admittedly and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.5: 0.5；The solid material is calculated as corn flour in mass ratio: bean cake powder: wheat bran=65: 25: 10；The component and weight ratio of the inorganic salts For：Potassium dihydrogen phosphate 15%, magnesium sulfate 5%, ammonium sulfate 15%, precipitated calcium carbonate 65%；The solid medium of the same step of bulk fermentation culture medium (2) in step (3).
- 6. the preparation method of biological control agent as claimed in claim 4, it is characterized in that, comprise the following steps：(1) spore of Bei Laisi Bacillus strains XT1-3 is transplanted to beef extract fluid nutrient medium, 27~29 DEG C of shaking table vibrations 3~5d of culture obtains seed liquor；(2) ratio for the seed liquor in mass ratio 10% for preparing step (1) is inoculated into solid medium, is shaken at 27~29 DEG C Swing 3~5d of culture；(3) culture of step (2) the culture ratio of sterile water in mass ratio 1: 15 is mixed, filtrate is pressed volume by filtering Ratio than 1: 6 is seeded to bulk fermentation culture medium, 27~29 DEG C of room temperature, relative humidity more than 85% fermenting cellar in ferment training Support 8~9d.
- 7. purposes of the biological control agent in watermelon blight is prevented described in claim 3.
- 8. it is a kind of for preventing the biological control method of watermelon blight, it is characterized in that, to the watermelon application power with droop Profit requires the biological control described in Bei Laisi bacillus (B.velezensis) strain X T1-3 or claim 3 described in 1 Preparation.
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