CN108396002A - One bacillus licheniformis and its application in preventing Muskmelon Fusarium wilt - Google Patents
One bacillus licheniformis and its application in preventing Muskmelon Fusarium wilt Download PDFInfo
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Abstract
Application the invention discloses a bacillus licheniformis and its in preventing Muskmelon Fusarium wilt.The bacterial strain is bacillus licheniformis (Bacillus licheniformis) bacterial strain LT4 3, it detaches and obtains from the river silt of Jining City in Shandong Province canal, it is preserved within 26th China Committee for Culture Collection of Microorganisms's common micro-organisms center in September in 2017, preserving number is CGMCC No.14745.Test result shows：3 wettable powders of lichem bacillus strain LT4 prevent Muskmelon Fusarium wilt significant effect, and control effect reaches 74.31%, and muskmelon grows fine, and no poisoning generates.
Description
Technical field
The invention belongs to technical field of plant disease biological control.Specifically, the present invention relates to one plant of lichens gemma bars
Bacterium and its application in preventing Muskmelon Fusarium wilt, and the biological control agent using bacillus licheniformis preparation.
Background technology
Muskmelon is also known as muskmelon, ripe melon, "Hami" melon etc., and in China, cultivation history is long, because muskmelon pulp nutritive value is higher,
Have the function of to relieve summer heat diuresis, protection liver, replenishing vitamins etc., along with looking good, smell good and taste good so that muskmelon is liked by masses deeply.
Muskmelon is as annual trailing plant, and worldwide cultivation, China's melon yield occupy first of various countries extensively.In recent years, with
The increase of Muskmelon Planting density and multiple crop index, the frequency that muskmelon pest and disease damage occurs is also higher and higher, wherein Muskmelon Fusarium wilt
It is then one of the Major Diseases for influencing muskmelon agricultural production.Muskmelon Fusarium wilt is also known as dead seedling, dead rattan, wilt disease, dead arm, open country
It can occur with Protectorate cultivation, be the upper important fungal disease of muskmelon production, often cause melon seedling withered death.Muskmelon Fusarium wilt
Caused by Fusarium oxysporum muskmelon specialized form (Fusarium oxysporum f.sp.melonis).Muskmelon Fusarium wilt is typical
Silborne fungal diseases, can fall ill from seedling stage to Adult plant.Wherein, the phase morbidity of bearing fruit of blooming is most heavy, often causes melon seedling withered
It is dead.
The main method of prevention Muskmelon Fusarium wilt or chemical prevention at present keep Muskmelon Fusarium wilt sick due to long-term administration
Opportunistic pathogen develops immunity to drugs, and eventually leads to that control effect is undesirable, along with a large amount of uses of chemical agent are to muskmelon quality safety
Many negative effects are caused with environment, and biological control has to safety of human and livestock, environment compatibility is good and pathogen is not likely to produce
The advantages that drug resistance, therefore the green non-pollution biological control of Muskmelon Fusarium wilt is more and more paid close attention to by people.
Bacillus licheniformis is bacterial strain in bacillus, is to be used widely and study at present, and dive with huge
In one of the bacterial strain of Development volue, thalline can generate antibacterial substance, have unique biology take oxygen mechanism, inhibit disease
The growth and breeding of opportunistic pathogen, while a variety of growth-promoting related substances can be generated, promote plant growth.
Invention content
The object of the present invention is to provide a kind of new microorganism --- lichens gemma for efficiently preventing Muskmelon Fusarium wilt
Bacillus LT4-3.The present invention lichem bacillus strain have the speed of growth is fast, yield is big, resistance is strong, plant (especially
Muskmelon) 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 lichens bud
Spore bacillus (Bacillus licheniformis) bacterial strain LT4-3, detaches from the river silt of Jining City in Shandong Province canal 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.14745.It is with following biological characteristics：It is cultivated in beef extract-peptone agar medium or LB (Luria-Bertani)
The bacterial strain shape is rod-shaped on base, and flagellum feature is apparent, and single bacterium colony is circle, and dirty white, opaque, central uplift, edge are whole
Together, there is fold in the later stage；It in beef extract-peptone agar medium, is cultivated at 28 DEG C two days later, microscopy, somatic cells are in bar
Shape can move.It is positive (being control with Escherichia coli) through Gram's staining.It is oval that gemma is observed after spore staining,
Middle life is close middle raw, embraces a capsule and does not expand.
The cultural method or propagation method of bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 of the present invention include：
(1) Nostoc commune Vanch, which preserves, uses LB culture mediums, is formulated and is：Tryptone 10g, yeast extract 5g, sodium chloride
10g, 15~20g of agar, distilled water 1000mL, pH are adjusted to 7.0~7.2.
(2) laboratory fluids culture uses LB liquid medium, is formulated and is：Tryptone 10g, yeast extract 5g, chlorine
Change sodium 10g, distilled water 1000mL, pH are adjusted to 7.0~7.2.
(3) solid culture based formulas：Including solid material and inorganic salts, the mass ratio of the solid material and inorganic salts is 98.6:
0.4；The solid material is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=54: 30: 10: 6；The component of the inorganic salts
And mass ratio is：Potassium dihydrogen phosphate 21%, magnesium sulfate 5%, ammonium sulfate 9%, precipitated calcium carbonate 65%.
(4) bulk fermentation culture formula：With solid culture based formulas in (3).
The present invention also provides a kind of biological control agent for preventing Muskmelon Fusarium wilt, the biological control agent packets
Include bacillus licheniformis (B.licheniformis) the bacterial strain LT4-3.
Also, the present invention also provides a kind of biological control method for preventing Muskmelon Fusarium wilt, the biological controls
Method includes applying above-mentioned bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 to the Muskmelon Plants with droop
Or above-mentioned biological control agent.
Present invention provides above-mentioned lichem bacillus strain LT4-3 or above-mentioned biological control agent in prevention muskmelon
Purposes in droop.
The preparation method of biological control agent of the present invention includes the following steps：
(1) seed liquor of the lichem bacillus strain LT4-3 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 27~29 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, 27~29 DEG C of room temperature, 85% or more relative humidity fermenting cellar in carry out fermented and cultured.
In one embodiment of the invention, the preparation method comprises the following steps：
(1) spore of the lichem bacillus strain LT4-3 is transplanted to LB liquid medium, 27~29 DEG C of shaking tables shake
It swings 3~5d of culture and obtains seed liquor；
(2) ratio of seed liquor in mass ratio 10% prepared by step (1) is inoculated into solid medium, 27~29 DEG C
3~5d of lower shaken cultivation；
(3) ratio of the culture sterile water in mass ratio 1: 15 of step (2) culture is mixed, 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, 85% or more relative humidity fermenting cellar in send out
8~9d of ferment culture.
Wherein, the LB liquid medium in step (1), is formulated and is：Tryptone 10g, yeast extract 5g, sodium chloride
10g, distilled water 1000mL, pH are adjusted to 7.0~7.2.
Solid medium in step (2) includes solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.6:
0.4；The solid material is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=54: 30: 10: 6；The component of the inorganic salts
And mass ratio is：Potassium dihydrogen phosphate 21%, magnesium sulfate 5%, ammonium sulfate 9%, precipitated calcium carbonate 65%.
The solid medium of the same step of bulk fermentation culture medium (2) in step (3).
Experiment show lichem bacillus strain of the invention have the speed of growth is fast, sporulation quantity is big, resistance is strong,
Plant (especially muskmelon) surface can rapid, high volume the features such as colonizing, therefore have a good application prospect.By the lichens bud
Biological control agent prepared by spore bacillus can not only efficiently prevent Muskmelon Fusarium wilt evil, moreover it is possible to muskmelon growth is effectively facilitated,
It is a kind of biological control agent of great application prospect.The microorganism formulation can be used as biological pesticide or bio-feritlizer, prevention
Muskmelon Fusarium wilt.Test result shows：Bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 wettable powders are prevented
Muskmelon Fusarium wilt significant effect, control effect reaches 74.31%, and muskmelon grows fine, and no poisoning generates.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are merely exemplary, and it is not intended to limit the scope of the present invention in any way.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are within the scope of the invention.
Embodiment 1
1, the isolation and purification of bacillus licheniformis (B.licheniformis) LT4-3
Bacillus licheniformis (B.licheniformis) LT4-3 of the present invention be from river silt using dilution-plate method and
Plate streak separation obtains, and separation method is：
(1) separation of bacillus
River silt sample was derived from Jining City in Shandong Province canal on May 15th, 2014.1g river silt is weighed in 100mL sterile waters
In, 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 100 μ L 10-2、
10-3、10-4Dilution is coated on LB culture medium flat plates, and each gradient is coated with three parallel, picking LB after culture 2d at 30 DEG C
The microbial bacteria of different shape on culture medium falls within and crosses on LB culture medium flat plates, and bacterium colony growing state is observed in timing.
Then plate streak is used, purifies Bacillus strain, and number preservation.
(2) screening of the efficient antagonistic strain of Muskmelon Fusarium wilt
1. primary dcreening operation：Using opposite culture method, PDA (Potato Dextrose Agar) tablet is prepared, with card punch in bud
Spore bacillus, Muskmelon Fusarium wilt pathogen edge take the bacteria cake of a diameter of 5mm, be implanted in respectively tablet it is opposite both sides center, 25
DEG C constant temperature incubation observes inhibiting effect of the bacillus to pathogen day by day.
2. secondary screening：The Bacillus strain with efficient antagonistic activity screened is subjected to secondary screening, is mainly passed through 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 bacterial strain LT4-3 obtained to screening is identified.
The present inventor obtains one plant of bacillus licheniformis that can efficiently prevent Muskmelon Fusarium wilt by a large amount of screening operations
(B.licheniformis)LT4-3.It is demonstrated experimentally that the bacillus licheniformis original powder shown in preventing Muskmelon Fusarium wilt it is non-
Normal efficient control effect so that muskmelon grows fine.Thus, bacillus licheniformis of the invention is with wide application prospect
New strain of Bacillus licheniformis, can be used for prepare prevention Muskmelon Fusarium wilt biological control agent.
2, bacterial strain is identified
(1) Microbiological Characteristics：The bacterial strain shape is rod-shaped in beef extract-peptone agar medium or LB culture mediums,
Flagellum feature is apparent, and single bacterium colony is circle, and there are fold in dirty white, opaque, central uplift, neat in edge, the later stage；In beef extract
It on peptone agar medium, is cultivated at 28 DEG C two days later, microscopy, somatic cells are in rod-shaped, can be moved.It is in through Gram's staining
Positive (being control with Escherichia coli).It is oval that gemma is observed after spore staining, middle life or close middle raw is embraced a capsule and do not expanded.
(2) molecular biological characteristic
The 16s rDNA gene sequencings result of the bacterial strain is following (SEQ-1)：
TCCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG
CGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACA
GATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTC
ATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACT
GAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACA
ACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGT
AAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTC
AGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACAC
TTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGT
CAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAAT
TCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAG
ACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTG
CTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCC
CTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGG
AAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTC
GGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAG
CCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCC
CAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCG。
(3) cellular morphology and physical and chemical experiment result
The cellular morphology and physical and chemical experiment result of 1 bacillus licheniformis of table (B.licheniformis) LT4-3
| 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 round | 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 | + |
The bacterial strain is accredited as bacillus licheniformis (Bacillus licheniformis), is protected within 26th in September in 2017
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.14745.
Embodiment 2
1, Bacillus licheniformis strain fermentation process
LB liquid medium formula is：Tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH
It is adjusted to 7.0~7.2.
Solid culture based formulas：Gu the mass ratio of material, inorganic salts and water, the solid material and inorganic salts is 98.6: 0.4；Institute
It states solid material and is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=54: 30: 10: 6；The component and quality of the inorganic salts
Than for：Potassium dihydrogen phosphate 21%, magnesium sulfate 5%, ammonium sulfate 9%, precipitated calcium carbonate 65%.Preparation method：Suitable water is added
(10-15 times of quality) dissolves inorganic salts, obtains inorganic salt solution；Inorganic salt solution is mixed with solid material, and water final adjustment is added
The water content of solid medium is 40-60%, and solid medium is held in the hand, has water in webs but not drippage is advisable.
The a large amount of solid fermentation process of bacillus licheniformis (B.licheniformis) bacterial strain LT4-3：
1. strain seed liquor culture
By a small amount of spore of lichem bacillus strain (B.licheniformis) LT4-3 pickings from test tube slant, move to
In LB liquid 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：
2. the culture sterile water of middle solid culture is pressed into 1: 15 dilution proportion, is used in combination sterile gauze to filter, removes thick
Slag, as produces bacterium solution, and inoculative proportion is inoculated in bulk fermentation culture medium by 1: 6.The raw material of inoculation is placed in (28 DEG C of fermenting cellar
With 85% or more relative humidity) in 8~9d of fermented and cultured；Then it is further dried and bacillus licheniformis is made
(B.licheniformis) bacterial strain LT4-3 original powders, active bacteria up to 15,000,000,000/gram.
4. wettable powder
Raw material proportioning：Bacillus licheniformis LT4-3 original powders 10%, glucose 45%, wetting agent 1,004 6%, dispersant
NNO 2.0%, CMC 1%, precipitated calcium carbonate surplus.
Prevention for clear bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 pulvis to Muskmelon Fusarium wilt
Effect provides foundation for its popularization and application.We test 2 months~March in 2016 in Jinan City, Shandong Province Zhangqiu area, existing
Test result is reported as follows：
1 material to be tested and method
1.1 reagent agent
Bacillus licheniformis (B.licheniformis) the bacterial strain LT4-3 powder produced by 2 fermentation process of the embodiment of the present invention
Agent (1,500,000,000/gram spore pulvis WP)；(Jin Liang Fine Chemical Co., Ltd of Henan Province produces 60% carbendazol wettable powder, city
It sells).
1.2 test materials and controlling object
Test material is muskmelon, and kind is capital sweet tea 5；Controlling object is Muskmelon Fusarium wilt (Fusarium oxysporum
f.sp.melonis)。
1.3 experimental field situations
Experimental field it is located at Jinan City, Shandong Province Zhangqiu area high-ranking official stockaded village town Muskmelon Planting base greenhouse.In recent years, this area by
In continuously plantation muskmelon, Muskmelon Fusarium wilt occurs commonplace.Experiment soil types be moisture soil, the content of organic matter 1.72%,
PH value is 7.5, and all experimental plot cultivation conditions and management measure are consistent, and Muskmelon Fusarium wilt is in early stage when dispenser.
1.4 experimental designs and arrangement
This experiment totally 3 processing, respectively 20 times of bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 pulvis
Liquid, 60% carbendazol wettable powder, 800 times of liquid, not dispenser clear water compare, and each handle 4 repetitions, each handle 140
Strain.In early stage to diseased plant and its neighbouring plant pouring root, 10 days primary, continuous 3 times, every time per diseased plant irrigation liquid 0.25kg.
In on 2 25th, 2016, March 5 and March 15 each pouring root it is primary.
1.5 pilot surveies and computational methods
1.5.1 meteorological condition
1st dispenser (the 25 days 2 months) same day is cloudy, 3 grades of wind-force, and the highest temperature is 9 DEG C, and the lowest temperature is -2 DEG C, relatively
Humidity is 51%.2nd dispenser (March 5), the same day is cloudy, 3 grades of wind-force, and the highest temperature is 13 DEG C, and the lowest temperature is 3 DEG C, phase
It is 50% to humidity；3rd dispenser (March 15) same day is fine, 3 grades of wind-force, and the highest temperature is 15 DEG C, and the lowest temperature is 5 DEG C, phase
It is 52% to humidity.
1.5.2 drug effect and security survey
Efficacy survey：14d is investigated after last time dispenser.120 plants of investigation, the whole blades of investigation, Investigate incidence,
Record disease index simultaneously calculates disease index.
Security survey：The safety of 7d and 14d observations muskmelon, 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.
Muskmelon Fusarium wilt grade scale：
0 grade：Complete stool is without illness.
1 grade：1/4 or less blade of plant shows wilting symptom.
2 grades：1/4~1/2 blade of plant shows wilting symptom.
3 grades：1/2 or more blade of plant shows wilting symptom.
4 grades：Plant is withered because of disease.
1.5.3 drug effect computational methods
Control effect is calculated by formula (1), (2)：
In formula：CK0--- disease index before blank district dispenser；
CK1--- disease index after blank district dispenser；
PT0————Disease index before dispenser before chemicals treatment；
PT1————Disease index after dispenser after chemicals treatment.
1.5.4 muskmelon is directly affected
Observing medicament, whether there is or not poisoning to muskmelon, record the type and extent of poisoning.In addition, also to record to muskmelon growing way
It influences.
Poisoning is recorded with following manner：
If (a) poisoning can be measured or calculate, absolute value representation, such as plant height are used.
(b) in other cases, it can estimate poisoning degree and frequency by following two methods：
1. the poisoning degree per cell is recorded according to poisoning stage division, with-,+, ++, +++, ++++it indicates.
Poisoning stage division：
—：Without poisoning；
+：Slight poisoning, does not influence plant growth；
++：Moderate poisoning, it is resilient, phytomass will not be reduced；
+++：Moderate poisoning, influences plant normal growth, and a degree of reduction is caused to phytomass；
++++：Serious poisoning, plant growth are obstructed, and phytomass reduces serious.
2. by chemicals treatment district compared with blank district, its poisoning percentage is evaluated.Meanwhile wanting accurate description plant
Symptom of chemical damage (dwarfing, chlorisis, deformity etc.).
2 results
Control effect of 2.1 reagent agents to Muskmelon Fusarium wilt
Reagent agent is to the control effect of Muskmelon Fusarium wilt as shown in table 2-3.Table 3 is the results show that bacillus licheniformis
(B.licheniformis) bacterial strain LT4-3 wettable powders and 60% carbendazol wettable powder all have Muskmelon Fusarium wilt
Preferable control effect, wherein the former control effect reach 70% or more.
From the point of view of the disease index of Muskmelon Fusarium wilt field trial (table 3), 14d after last time dispenser, lichens gemma bar
Bacterium (B.licheniformis) LT4-3 control effects are 74.31%, hence it is evident that are higher than 60% carbendazol wettable powder 55.42%
Control effect.
Muskmelon Fusarium wilt disease incidence before 2 three kinds of chemicals treatments of table
Control effect of the 3 three kinds of chemicals treatments of table to Muskmelon Fusarium wilt
2.2 muskmelon security surveys
It is observed through dispenser 7d and 14d, each chemicals treatment district is compared with check plot, and muskmelon growth is normal, and no poisoning generates, and says
Bright bacillus licheniformis pulvis is to muskmelon safety.
3 brief summaries
3.1 from the point of view of disease index and control effect, and bacillus licheniformis (B.licheniformis) bacterial strain LT4-3 (is protected
Tibetan CGMCC No.14745) there is preferable control effect, its control effect of 14d after last time dispenser to Muskmelon Fusarium wilt
Reach 70% or more.
3.2 in terms of muskmelon growing way, and bacillus licheniformis (B.licheniformis) bacterial strain LT4-3, which has muskmelon, to be promoted
Growth result, without poisoning, securely and reliably.
SEQUENCE LISTING
<110>Shandong new standing grain rich crop nutrition Co., Ltd
<120>One bacillus licheniformis and its application in preventing Muskmelon Fusarium wilt
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1381
<212> DNA
<213>Bacillus licheniformis（B. licheniformis）The 16s rDNA gene orders of bacterial strain LT4-3
<400> 1
tcctcaccga cttcgggtgt tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc 60
cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca gcttcacgca 120
gtcgagttgc agactgcgat ccgaactgag aacagatttg tgggattggc ttaacctcgc 180
ggtttcgctg ccctttgttc tgtccattgt agcacgtgtg tagcccaggt cataaggggc 240
atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc accttagagt 300
gcccaactga atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa 360
catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgcc cccgaagggg 420
acgtcctatc tctaggattg tcagaggatg tcaagacctg gtaaggttct tcgcgttgct 480
tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca 540
gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actaaggggc 600
ggaaaccccc taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc 660
ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga gtcgccttcg 720
ccactggtgt tcctccacat ctctacgcat ttcaccgcta cacgtggaat tccactctcc 780
tcttctgcac tcaagttccc cagtttccaa tgaccctccc cggttgagcc gggggctttc 840
acatcagact taagaaaccg cctgcgagcc ctttacgccc aataattccg gacaacgctt 900
gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta 960
ccgtcaaggt gccgccctat ttgaacggca cttgttcttc cctaacaaca gagctttacg 1020
atccgaaaac cttcatcact cacgcggcgt tgctccgtca gactttcgtc cattgcggaa 1080
gattccctac tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga 1140
tcaccctctc aggtcggcta cgcatcgtcg ccttggtgag ccgttacctc accaactagc 1200
taatgcgccg cgggtccatc tgtaagtggt agccgaagcc accttttatg tctgaaccat 1260
gcggttcaga caaccatccg gtattagccc cggtttcccg gagttatccc agtcttacag 1320
gcaggttacc cacgtgttac tcacccgtcc gccgctaaca tcagggagca agctcccatc 1380
g 1381
Claims (8)
1. the deposit number of bacillus licheniformis (Bacillus licheniformis) bacterial strain LT4-3, the bacterial strain are CGMCC
No.14745。
2. purposes of the lichem bacillus strain LT4-3 described in claim 1 in preventing Muskmelon Fusarium wilt.
3. a kind of biological control agent, including lichem bacillus strain LT4-3 described in claim 1.
4. the preparation method of the biological control agent described in claim 3, characterized in that include the following steps：
(1) seed liquor of lichem bacillus strain LT4-3 is prepared；
(2) seed liquor prepared by step (1) is inoculated into solid medium, constant temperature incubation at 27~29 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,
27~29 DEG C of room temperature, 85% or more relative humidity fermenting cellar in carry out fermented and cultured.
5. the preparation method of biological control agent as claimed in claim 4, characterized in that
Solid medium in the step (2) includes solid material and inorganic salts, and the mass ratio of the solid material and inorganic salts is 98.6:
0.4；The solid material is calculated as rice husk in mass ratio: corn flour: bean cake powder: wheat bran=54: 30: 10: 6；The component of the inorganic salts
And mass ratio is：Potassium dihydrogen phosphate 21%, magnesium sulfate 5%, ammonium sulfate 9%, 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 the biological control agent described in claim 5, characterized in that include the following steps：
(1) spore of lichem bacillus strain LT4-3 is transplanted to LB liquid medium, 27~29 DEG C of shaking table shaken cultivations 3
~5d obtains seed liquor；
(2) ratio of seed liquor in mass ratio 10% prepared by step (1) is inoculated into solid medium, is shaken at 27~29 DEG C
Swing 3~5d of culture；
(3) ratio of the culture sterile water in mass ratio 1: 15 of step (2) culture is mixed, filtering, filtrate is pressed into volume
Ratio than 1: 6 is seeded to bulk fermentation culture medium, 27~29 DEG C of room temperature, 85% or more relative humidity fermenting cellar in ferment training
Support 8~9d.
7. purposes of the biological control agent in preventing Muskmelon Fusarium wilt described in claim 3.
8. a kind of biological control method for preventing Muskmelon Fusarium wilt, characterized in that applied to the Muskmelon Plants with droop
With the biological control agent described in lichem bacillus strain LT4-3 described in claim 1 or claim 3.
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| CN110157636A (en) * | 2019-04-01 | 2019-08-23 | 成都市农林科学院 | A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis |
| WO2022127125A1 (en) * | 2020-12-16 | 2022-06-23 | 中国热带农业科学院环境与植物保护研究所 | Slow-release special fertilizer for muskmelons and preparation method therefor |
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| CN110157636A (en) * | 2019-04-01 | 2019-08-23 | 成都市农林科学院 | A kind of bacillus licheniformis, screening technique, the feed using and containing the bacillus licheniformis |
| WO2022127125A1 (en) * | 2020-12-16 | 2022-06-23 | 中国热带农业科学院环境与植物保护研究所 | Slow-release special fertilizer for muskmelons and preparation method therefor |
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| CN108396002B (en) | 2019-12-06 |
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