CN108913618A - A kind of bacillus amyloliquefaciens JSPB14 and its application - Google Patents
A kind of bacillus amyloliquefaciens JSPB14 and its application Download PDFInfo
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- CN108913618A CN108913618A CN201810739553.7A CN201810739553A CN108913618A CN 108913618 A CN108913618 A CN 108913618A CN 201810739553 A CN201810739553 A CN 201810739553A CN 108913618 A CN108913618 A CN 108913618A
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- 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
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- 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
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Abstract
The invention belongs to crop disease technical field of biological control, in particular to a kind of bacillus amyloliquefaciens JSPB14 and its application, the bacillus amyloliquefaciens are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 20th, 2017, and culture presevation number is CGMCC No.14934；Higher antagonistic activity is all had to fester germ, xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria of rear Pleurotus eryngii is adopted, biocontrol agent using bacterial strain of the present invention preparation is to adopting rear Pleurotus eryngii ulcer disease and bacterial leaf streak of rice shows higher biocontrol effect, the residual of chemical pesticide and farm antibiotics can be avoided or reduced, food and Environmental security are improved, there is preferable economic and social benefit.
Description
Technical field
The invention belongs to crop disease technical field of biological control, and in particular, to a kind of bacillus amyloliquefaciens
JSPB14 and its application.
Background technique
Pleurotus eryngii (Pleurotus eryngii) belong to Agaricales (Aaricales) edible fungus, nutrition with higher and
Medical value.Currently, the yield of China's Pleurotus eryngii is up to 500,000 tons/year or more, annual value of production 45~6,000,000,000 yuan.Rear Pleurotus eryngii is adopted to fester
Disease is to infect caused bacteriosis by Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis),
General disease incidence, up to 50% or more when serious, causes huge economic losses 15%~30%.Due to adopting rear fruits and vegetables fungicide
Higher using standard, the fungicide type and dosage that can be selected are severely limited, and most countries are forbidden
It adopts on rear fruits and vegetables using farm antibiotics, causes alternative medicament kind seldom, control efficiency is undesirable.Low temperature (1 DEG C~
5 DEG C) be the effective measures for controlling the disease, still, the whole cold chain control after adopting to dining table need advanced technical equipment and
Expensive logistics cost, significantly reduces the economic benefit of Pleurotus eryngii industry.
Rice bacteriosis mainly includes bacterial leaf streak of rice (Xanthomonas oryzae
It pv.oryzicola) and bacterial blight of rice (Xanthomonas oryzae pv.oryzae), is in China's Rice Production
Important disease, 800~15,000,000 mu of long-term occurring area, hazard area causes Rice Yield Loss Caused 5% up to 300~5,000,000 mu
~10%, up to 50% or more, or even total crop failure when serious, become one of the important hidden danger of China's rice safety in production.Currently, rice
The prevention and control of bacteriosis rely primarily on chemical pesticide and farm antibiotics.However, chemical pesticide and antibiotic is long-term unreasonable
Using causing pathogen drug resistance to gradually increase, administration dosage blindly increases, and control efficiency is undesirable, while remains of pesticide is to food
Product safety and ecological environment constitute a serious threat.
Develop safe and efficient, novel chemical bactericide with high costs, investment risk is huge.Biology based on microorganism
Control Technology is considered as one of following control crop bacterium disease the main direction of development.Bacillus (Bacillus spp.)
Belong to gram-positive bacterium, be distributed widely in the various habitats such as soil, plant surface, water body, agricultural wastes, has wide
The antibacterial activity of spectrum, higher gemma yield and stronger adverse circumstance adaptability, it is considered to be most application and development potentiality has
One of beneficial microorganism.Wherein, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) be it is a kind of by scientist and
The Biocontrol microorganism safe to the human body that consumer is widely recognized as, has had multiple bacterial strains to be used for prevention and control plant disease.But
There are notable differences for biological characteristics, antimicrobial spectrum of separate sources bacillus amyloliquefaciens bacterial strain etc., have strain specificity.
Summary of the invention
The present invention solves the above-mentioned technical problems in the prior art, provide a kind of bacillus amyloliquefaciens JSPB14 and
It is applied.
To solve the above problems, technical scheme is as follows：
The present invention provides a kind of bacillus amyloliquefaciens JSPB14, is deposited in China Microbiological on November 20th, 2017
Culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, postcode 100101), classification naming is Bacillus amyloliquefaciens, strain
Deposit number is CGMCC No.14934.The Bacillus amyloliquefaciens strain is to separate to obtain from the cultivation matrix of Pleurotus eryngii
, which is accredited as by the method combined using colony morphological observation, thalli morphology observation and gyrB gene sequencing
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and it is named as JSPB14.
Bacillus amyloliquefaciens strain JSPB14 cultivates the bacterium after 48h in 2YT solid medium tablets, under the conditions of 30 DEG C
Fall feature：Single colonie is milky, flat, round or subcircular, diameter 2.5mm~4.0mm, the shrinkage of bacterium colony surface, edge saw
Dentation；Its thalli morphology be it is rod-shaped, about 0.5 μm of diameter, about 1 μm~2 μm of length, gemma be in silk cocoon shape.Used 2YT solid
Medium component is：Tryptone 17g, yeast extract 10g, NaCl 5g, agar 15g, distilled water are settled to 1000mL, pH
Sterilize 20min under the conditions of 7.0,121 DEG C.
The present invention provides the tunning of the bacillus amyloliquefaciens JSPB14.
The present invention is provided to be produced comprising the bacillus amyloliquefaciens JSPB14 and/or bacillus amyloliquefaciens JSPB14 fermentation
The biocontrol agent of object.
The present invention is provided to be produced comprising the bacillus amyloliquefaciens JSPB14 and/or bacillus amyloliquefaciens JSPB14 fermentation
The preparation method of the biocontrol agent of object：
Preparation method one
(1) activation culture：Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, streak inoculation is to fresh
On 2YT solid plate, for 24 hours~36h is cultivated under the conditions of 30 DEG C；
(2) prepared by seed liquor：In picking one single colonie inoculation 2YT fluid nutrient medium, shaken cultivation 16h at 30 DEG C~
36h, frequency of oscillation are 100r/min~200r/min；
(3) preparation of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution：Step (2) resulting seed liquor is inoculated into the ratio column of 1%~5% (v/v)
It ferments in Landy culture medium, fermentation temperature is 26 DEG C~31 DEG C, and the initial pH of culture medium is 6.5~7.2, with ventilation ratio
0.5:1 to 2.5:Fermentation ventilation is carried out between 1, mixing speed is 75r/min~200r/min, and fermentation time is 36h~48h；
(4) preparation of biocontrol agent A：The biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution centrifugation that will be fermented in above-mentioned steps (3), takes sterile supernatant, adds
Potassium sorbate, then sterile filling, room temperature preservation.
Preferably, in the step (4), the final concentration of 1g/L of potassium sorbate is added.
Preparation method two
(1) activation culture：Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, streak inoculation is to fresh
On 2YT solid plate, for 24 hours~36h is cultivated under the conditions of 30 DEG C；
(2) prepared by seed liquor：In picking one single colonie inoculation 2YT fluid nutrient medium, shaken cultivation 16h at 30 DEG C~
36h, frequency of oscillation are 100r/min~200r/min；
(3) preparation of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution：Step (2) resulting seed liquor is inoculated into the ratio column of 1%~5% (v/v)
It ferments in Landy culture medium, fermentation temperature is 26 DEG C~31 DEG C, and the initial pH of culture medium is 6.5~7.2, with ventilation ratio
0.5:1 to 2.5:Fermentation ventilation is carried out between 1, mixing speed is 75r/min~200r/min, and fermentation time is 36h~48h；
(4) preparation of biocontrol agent B：Addition cocoyl is esterified compound in the fermentation liquid prepared in above-mentioned steps (3)
And sodium benzoate, pH value is adjusted to 4.5 or so, then sterile filling, room temperature preservation.
Preferably, final concentration of 8% (v/v) of the esterification of cocoyl described in the step (4) compound, sodium benzoate
Final concentration of 1g/L.
Above-mentioned 2YT nutrient media components are：Tryptone 17g, yeast extract 10g, NaCl 5g, distilled water are settled to
Sterilize 20min under the conditions of 7.0,121 DEG C of 1000mL, pH.
Above-mentioned 2YT solid medium group is divided into：Tryptone 17g, yeast extract 10g, NaCl 5g, agar 15g, distillation
Water is settled to 1000mL, and sterilize 20min under the conditions of 7.0,121 DEG C of pH.
Above-mentioned Landy nutrient media components are：Pidolidone 5g/L, yeast extract 1g/L, L-phenylalanine 2mg/L, grape
Sugared 20g/L, KH2PO4 1g/L、KCI 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O 0.15g/L、CuSO4·
5H2O 0.16mg/L、MnSO4Sterilize 20min under the conditions of 6.8~7.2,121 DEG C of 5mg/L, pH.
The present invention provides the bacillus amyloliquefaciens JSPB14 or its tunning or the biocontrol agent is adopted in prevention and treatment
Application in Pleurotus eryngii ulcer disease and bacterial leaf streak of rice afterwards.
Application method of the biocontrol agent A after prevention and treatment is adopted in Pleurotus eryngii ulcer disease：Biocontrol agent A is diluted 1~5
Times, it is handled using immersion way and adopts rear Pleurotus eryngii, placed after drying according to adopting rear shelf conditions.
Application method of the biocontrol agent B in prevention and treatment bacterial leaf streak of rice：Biocontrol agent B is diluted 60~80
Times, even spraying is carried out in the early period of origination of rice bacterial disease cecospora spot, 7~9d of interval is sprayed 1 time again.
Compared with the existing technology, advantages of the present invention is as follows,
Bacillus amyloliquefaciens strain JSPB14 provided by the invention is a kind of newfound bacillus biological and ecological methods to prevent plant disease, pests, and erosion resource, right
It adopts fester germ, xanthomonas oryzae pv. oryzicola and rice leaf spot bacteria of rear Pleurotus eryngii and all has higher antagonistic activity, benefit
The biocontrol agent prepared with bacterial strain of the present invention is to adopting rear Pleurotus eryngii ulcer disease and bacterial leaf streak of rice shows higher life
Anti- effect can avoid or reduce the residual of chemical pesticide and farm antibiotics, improve food and Environmental security, have preferable
Economic and social benefit.
Detailed description of the invention
Fig. 1 is that the biocontrol microorganisms of 4 plants of Preliminary Identifications fester the antagonistic activity of germ to Pleurotus eryngii；
Fig. 2 is bacterium colony, thallus and brood cell form of the bacillus amyloliquefaciens JSPB14 on 2TY culture medium；
Fig. 3 is the phylogenetic analysis of the gyrB gene order based on bacillus amyloliquefaciens JSPB14；
Fig. 4 is growth and sporulation dynamic of the bacillus amyloliquefaciens JSPB14 in Landy culture medium；
Fig. 5 is the antibacterial activity of bacillus amyloliquefaciens JSPB14 different fermentations time secondary metabolite；
The antagonism that Fig. 6 is bacillus amyloliquefaciens JSPB14 composes measurement；
Fig. 7 is antagonistic activity of the bacillus amyloliquefaciens JSPB14 to Different Kinds of Pathogens bacterium；
Fig. 8 is prevention and treatment of the bacillus amyloliquefaciens JSPB14 biocontrol agent to rear apricot Bao ulcer disease is adopted；
Fig. 9 is bacillus amyloliquefaciens JSPB14 biocontrol agent to the control efficiency for adopting rear apricot Bao ulcer disease.
Specific embodiment
It is micro- to be preserved in China on November 20th, 2017 by Bacillus amyloliquefaciens strain JSPB14 according to the present invention
Biological inoculum preservation administration committee common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica, postcode 100101), classification naming is Bacillus amyloliquefaciens,
Culture presevation number is CGMCC No.14934.Rear Pleurotus eryngii is adopted used in the present invention to fester germ Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis) is that fall ill after adopting separation on Pleurotus eryngii, the identification of this research department obtains；Water
Rice bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae), xanthomonas oryzae pv. oryzicola (Xanthomonas
Oryzae pv.oryzicola), Xanthomonas campestris pv campestris pvs oryzae and oryzicola (Xanthomonas campestris
Pv.campestris), carrot soft rot Erwinia carrot subspecies (Erwinia carotovora
Subsp.carotovora), Ralstonia solanacearum (Ralstonia solanacearum), staphylococcus aureus
(Staphylococcus aureus) is that this research department saves bacterial strain, and other unit or individual can ask for this research department
Above 7 kinds of pathogenetic bacterias.
The following examples are intended to illustrate the invention, but is not limited to the present invention.Unless otherwise noted, in following embodiments
Technical method used is conventional method；Unless otherwise noted, experimental material used in following embodiments is conventional chemical examination
Agent and biochemical reagents.
2YT nutrient media components as described below are：Tryptone 17g, yeast extract 10g, NaCl 5g, distilled water constant volume
To 1000mL, sterilize 20min under the conditions of 7.0,121 DEG C of pH.2YT solid medium adds agar 15g/L.
Landy nutrient media components as described below are：Pidolidone 5g/L, yeast extract 1g/L, L-phenylalanine 2mg/
L, glucose 20g/L, KH2PO4 1g/L、KCI 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O 0.15g/L、
CuSO4·5H2O 0.16mg/L、MnSO4Sterilize 20min under the conditions of 6.8~7.2,121 DEG C of 5mg/L, pH.
MRS nutrient media components as described below are：Peptone 10g/L, yeast extract 5g/L, dibasic ammonium citrate 2g/L,
Glucose 20g/L, beef extract 10g/L, MgSO4.7H2O 0.58g/L、MnSO4.4H2O 0.25g/L、K2HPO42g/L, second
Sour sodium 2g/L, sterilize under the conditions of 6.8,121 DEG C of Tween 80 1mL/L, pH 20min.MRS solid medium adds agar 15g/L.
The product name " SF-98B " of " cocoyl esterification compound " as described below, from Changzhou, profit source peat product is limited
Company's purchase).
Embodiment 1：
The separation and identification of bacillus amyloliquefaciens bacterial strain JSPB14
(1) separation of microorganism resource to be screened：It is random in the rear higher cultivation matrix of Pleurotus eryngii ulcer disease disease incidence from adopting
Sample 50g, take 5 parts of matrix samples altogether, be uniformly mixed, take 10g matrix be put into containing 20 micro glass beads and 100mL sterilizing go from
In the 250mL conical flask of sub- water, it is placed in 150r/min on shaking table, shake culture 6h under the conditions of 30 DEG C.10min is stood, supernatant is taken
1mL draws each concentration gradient dilution Tu cloth 2YT solid plate of 100 μ L using 10 times of gradient dilutions of sterile deionized water,
Stationary culture 36h under the conditions of 30 DEG C.It is crossed and is purified 2 times on 2YT solid medium using oese picking single colonie, will purified
Different shape single colonie be transferred on 2YT solid plate, stationary culture 36h under the conditions of 30 DEG C, be stored in 4 DEG C it is spare.
(2) the plate antagonism screening of biocontrol bacterial strain：Each bacterial strain purified on 2YT solid plate will be stored in 2YT liquid
It is cultivated in culture medium, condition of culture 150r/min, 30 DEG C, 36h are spare as seed liquor；In addition, by Lactococcus lactis
In the glycerol strain streak inoculation to MRS solid plate of sp. strain SLPE1-3,36h is cultivated under the conditions of 30 DEG C, then picking list
Into the 250mL conical flask containing 50mL MRS fluid nutrient medium, stationary culture 12h under the conditions of 30 DEG C will be cultivated colony inoculation
Liquid (OD600≈ 1.0) it is added in the low temperature 2YT agar medium of liquid, it mixes rapidly, prepares 2YT Solid nutritional plate；It will
On strain to be tested seed liquor 2 μ L point to the 2YT Solid nutritional plate of preparation, each 4 strains to be tested of plate point；Each bacterial strain 3
A repetition；The antagonism plate handled well is placed under the conditions of 30 DEG C and cultivates 48h, bacterium to be determined is assessed by the diameter of antagonism circle
Antagonistic activity of the strain to Lactococcus lactis subsp. lactis.As a result, it has been found that in 367 bacterial strains of measurement, number JSPB3,
The antagonism loop diameter that JSPB14, JSPB106 and JSPB247 are generated is greater than 10mm, and wherein the antagonism loop diameter of JSPB14 reaches
13.0mm as shown in Figure 1.
(3) morphologic observation of biocontrol bacterial strain JSPB14：JSPB14 is diluted into coated plate culture on 2YT solid plate, is cultivated
Condition is 30 DEG C, 48h, and single colonie is milky, flat, round or subcircular, diameter 2.5mm~4.0mm, bacterium colony dry tack free
Shrinkage, edge sawtooth shape, as shown in Figure 2 A；Observed under scanning electron microscope, thalli morphology be it is rod-shaped, about 0.5 μm of diameter, length is about
1 μm~2 μm, gemma is in silk cocoon shape, as illustrated by figures 2 b and 2 c.
(4) Molecular Identification of biocontrol bacterial strain JSPB14：It is fallen within using sterilizing toothpick picking JSPB14 single bacterium containing 50mL
In the 250mL conical flask of 2YT fluid nutrient medium, 36h 150r/min, is cultivated under the conditions of 30 DEG C, and bacterial genomes DNA is utilized to extract
The total genomic dna of kit extraction JSPB14.Utilize the universal primer UP-1 (5 '-of bacterium gyrB gene
GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3 ') and UP-2r (5 '-
AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3 ') pcr amplified DNA segment；Utilize sequencing primer
UP-1S (5 '-GAAGTCATCATGACCGTTCTGCA-3 ') and UP-2Sr (5 '-AGCAGGGTACGGATGTGCGAGCC-3 ') are right
PCR product is sequenced.GyrB gene order and GenBank database based on acquisition, using 1.83 software of ClustalX and
5.05 software of MEGA carries out phylogenetic analysis to JSPB14, determines that the bacterial strain belongs to bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens), as shown in Figure 3.
Embodiment 2：
The antibacterial activity of Growth trends and secondary metabolite of the JSPB14 in Landy culture medium
From the JSPB14 bacterial strain of -70 DEG C of ultra low temperature freezers taking-up glycerol preservations, streak inoculation to 2YT solid plate, 30
36h is cultivated under the conditions of DEG C；It is inoculated into using sterilizing toothpick picking JSPB14 single colonie containing 50mL 2YT fluid nutrient medium
In 250mL conical flask, 36h 150r/min, is cultivated under the conditions of 30 DEG C, as seed liquor；By seed liquor according to the ratio of 5% (v/v)
Example is inoculated into the 250mL conical flask containing 50mL Landy fluid nutrient medium, 75r/min, cultivates 60h under the conditions of 30 DEG C.Point
Not after inoculation 0h, 4h, 8h, 12h, 18h, for 24 hours, 36h, 48h, 60h investigate thallus population density, specific method：Respectively upper
Point in time sampling is stated, carries out 10 times of gradient dilutions using aqua sterilisa, it is solid to draw each concentration gradient dilution coating 2YT of 100 μ L
Body plate, culture 48h carries out bacterium colony counting under the conditions of 30 DEG C.After inoculation 0h, 4h, 8h, 12h, 18h, for 24 hours, 36h,
48h, 60h investigate sporulation density, specific method：Respectively at above-mentioned point in time sampling, 80 DEG C of hot bath 30min are then sharp
10 times of gradient dilutions are carried out with aqua sterilisa, each concentration gradient dilution of 100 μ L is drawn and is coated with 2YT solid plate, 30 DEG C of conditions
Lower culture 48h carries out bacterium colony counting.As a result, it has been found that cell density reaches 3.87 × 10 when 12h after inoculation8Bacterium when CFU/mL, 48h
Volume density is most up to 9.13 × 108CFU/mL, as shown in Figure 4；12h starts largely to form brood cell after inoculation, for 24 hours when brood cell's density reach
To 8.07 × 107Brood cell's density reaches highest 1.47 × 10 when CFU/mL, 48h8CFU/mL, as shown in Figure 4.Each processing three
It repeats.
The fermented and cultured JSPB14 in Landy culture medium according to the method described above, after inoculation for 24 hours, 36h, 48h investigation
The antibacterial activity of fermentation secondary metabolite, specific method：Respectively at every bottle of sampling 2mL of above-mentioned time point, 15,000g centrifugations
10min, using 0.22 μm of sterilizing filter filtration sterilization, bacteria-free filtrate be stored in -70 DEG C it is spare.By Lactococcus lactis Asia
Kind bacterial strain SLPE1-3 culture solution (OD600≈ 1.0) it is added to the low temperature 2YT agar medium of liquid according to the ratio of 1% (v/v)
In, it mixes rapidly, prepares 2YT Solid nutritional plate；After plate solidification, 5 are uniformly made a call to using sterilization punchers on plate
40 μ L of aqua sterilisa (processing number is CK, similarly hereinafter), 100 μ g/mL ampicillin, 40 μ L are added in hole, each hole respectively
(Amp100), 40 μ L (for 24 hours) of without fermented liquid, 40 μ L (36h) of 36h without fermented liquid, 40 μ L of 48h without fermented liquid for 24 hours
Plate is placed under the conditions of 30 DEG C and cultivates 36h by (48h), investigates antagonism loop diameter.As a result, it has been found that with the increasing of fermentation time
Add, the antibacterial activity enhancing of sterile fermentation supernatant, antibacterial circle diameter of the sterile fermentation supernatant of 48h to Lactococcus lactis subsp. lactis
Reach the antagonistic activity (20.1mm) when 21.8mm, noticeably greater than 36h, it is living with the antibacterial of 100 μ g/mL ammonia benzyl mould of comparison medicament
Property (21.5mm) is not significantly different, as shown in Figure 5.
Embodiment 3：
The antibacterium of biocontrol bacterial strain JSPB14 composes measurement
In order to assess the antibacterium spectrum of the secondary antibacterial material of bacillus amyloliquefaciens bacterial strain JSPB14, selected 6 kinds it is main
Crop pathogenetic bacteria and a kind of human body opportunism pathogenetic bacteria carried out plate antagonistic experiment, including Lactococcus lactis
Subspecies (Lactococcus lactis subsp.lactis), rice leaf spot bacteria (Xanthomonas oryzae
Pv.oryzae), xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola), xanthomonas campestris
Sarson pvs oryzae and oryzicola (Xanthomonas campestris pv.campestris), carrot soft rot Erwinia carrot are sub-
Kind (Erwinia carotovora subsp.carotovora), Ralstonia solanacearum (Ralstonia solanacearum)
With staphylococcus aureus (Staphylococcus aureus).Specific method：Above-mentioned remove is taken out from -70 DEG C of ultra low temperature freezers
The glycerol strain of 6 kinds of pathogenetic bacterias except Lactococcus lactis subsp. lactis, crosses on 2YT solid medium respectively, and 30 DEG C
Cultivate 36h, pick them separately single bacterium and fall in 2YT fluid nutrient medium, 30 DEG C, under the conditions of 150r/min culture to OD600≈ 0.5, it is standby
With；The glycerol strain that Lactococcus lactis subsp. lactis is taken out from -70 DEG C of ultra low temperature freezers, crosses on MRS solid medium, and 30
36h is cultivated under the conditions of DEG C, picking single colonie is inoculated into MRS fluid nutrient medium, and stationary culture is to OD under the conditions of 30 DEG C600≈
0.5, it is spare；By the pathogen seed liquor of above-mentioned culture, the low temperature 2YT fine jade of liquid is respectively added to according to the ratio of 1% (v/v)
It in rouge culture medium, mixes rapidly, prepares 2YT Solid nutritional plate；It is equal using sterilization punchers on plate after plate solidification
It is even to make a call to 3 holes, it is spare.
The sterile fermented supernatant fluid of 36h and 48h of JSPB14 is prepared according to the description in embodiment 2, and 40 μ L sterilize respectively
Water (clear water compares CK), the 40 sterile fermented supernatant fluids of μ L 48h (processing 1), the 40 sterile fermented supernatant fluids of μ L 36h (processing 2) drop
To in the 2YT solid plate hole containing pathogenetic bacteria of preparation, plate is placed under the conditions of 30 DEG C and cultivates 36h, investigates antagonism
Loop diameter calculates antagonism circle radius (the radius 3mm for subtracting hole).As a result, it has been found that the secondary antibacterial substance of JSPB14 is not only to adopting
The Pleurotus eryngii pathogenetic bacteria Lactococcus lactis subsp. lactis that festers shows stronger antagonistic activity afterwards, to rice leaf spot bacteria,
Xanthomonas oryzae pv. oryzicola and Xanthomonas campestris pv campestris pvs oryzae and oryzicola also show stronger antagonistic activity, especially
17.1mm (36h) and 18.4mm (48h) are up to the inhibition zone radius of xanthomonas oryzae pv. oryzicola, as shown in Figure 6 and Figure 7.
Embodiment 4：
The preparation of JSPB14 biological prevention and control agent A and its prevention and treatment to rear apricot Bao ulcer disease is adopted
Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, in streak inoculation to 2YT solid plate, 30
It is cultivated for 24 hours under the conditions of DEG C；It is inoculated into using sterilizing toothpick picking JSPB14 single colonie containing 50mL 2YT fluid nutrient medium
In 250mL conical flask, 36h 100r/min, is cultivated under the conditions of 30 DEG C, as seed liquor；Seed liquor is arranged with the ratio of 1% (v/v)
It is inoculated in Landy culture medium and ferments, fermentation temperature is 26 DEG C, and the initial pH of culture medium is 6.5, with ventilation ratio 0.5:1
Fermentation ventilation is carried out, wherein ventilation ratio：Refer to per minute through the ratio between the volume of air of fermentation liquid and fermentating liquid volume；Mixing speed
For 75r/min, fermentation time 48h.By fermentation liquid 6, it is centrifuged 20min under the conditions of 000r/min, takes sterile supernatant, addition is dense eventually
Degree is the potassium sorbate of 1g/L, as JSPB14 biocontrol agent A, then sterile filling, room temperature preservation.
Application method of the biocontrol agent A after prevention and treatment is adopted in Pleurotus eryngii ulcer disease：Biocontrol agent A is diluted 1~5 times, benefit
It is handled with immersion way and adopts rear Pleurotus eryngii, placed after drying according to adopting rear shelf conditions, good control efficiency can be obtained.
Application case：
Picking Lactococcus lactis subsp. lactis bacterial strain SLPE1-3 single bacterium is fallen within containing 50mL MRS fluid nutrient medium
In 250mL conical flask, stationary culture 16h under the conditions of 30 DEG C is spare as cause of disease inoculum.Select uniform apricot Bao
Mushroom, using sterilizing scalpel, in Pleurotus eryngii mushroom handle middle and lower part mark signature wound, wound size is about 2cm × 2cm, wound depth
About 1mm.Total 3 processing：(1) biological and ecological methods to prevent plant disease, pests, and erosion is handled, and the 20 μ L of Lactococcus lactis subsp. lactis inoculum of preparation is inoculated into apricot Bao
On mushroom wound, it is stored at room temperature 30min, then impregnates 30s in 2 times of dilutions of biocontrol agent of above method preparation respectively, is located
Reason number is JSPB14-S+SLPE1-3；(2) 20 μ L aqua sterilisas are added dropwise in wound in clear water control treatment, and sterile water impregnates 30s,
Processing number is CK；(3) it is individually inoculated with the control treatment of pathogen SLPE1-3, the Lactococcus lactis subsp. lactis of preparation is connect
Kind 20 μ L of body is inoculated on wound, and sterile water impregnates 30s, and processing number is SLPE1-3.The Pleurotus eryngii of each immersion treatment is pulled out
It dries, is then placed in plastics antistaling box, inoculation wound is upward.Antistaling box is sealed, 15 DEG C of constant incubators are placed on
Then the temperature of constant incubator is turned up to 20 DEG C, continues to be incubated for by interior incubation 4d, the 8d and 12d investigation after processing
The disease incidence of Pleurotus eryngii.As a result as shown in Figure 8 and Figure 9, the disease incidence for being only inoculated with the control treatment Pleurotus eryngii of pathogen is up to
88.9% (8d) and 97.2% (12d)；The Pleurotus eryngii disease incidence of biological prevention and control agent processing is respectively 0 (8d) and 5.6% (the
12d), control efficiency is excellent.
Embodiment 5：
The preparation of JSPB14 biocontrol agent B and its prevention and treatment to bacterial leaf streak of rice
Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, in streak inoculation to 2YT solid plate, 30
36h is cultivated under the conditions of DEG C；It is inoculated into 1 containing 200mL 2YT fluid nutrient medium using sterilizing toothpick picking JSPB14 single colonie,
In 000mL conical flask, 16h 200r/min, is cultivated under the conditions of 30 DEG C, as seed liquor；Seed liquor is arranged with the ratio of 5% (v/v)
It is inoculated into Landy culture medium and ferments, fermentation temperature is 31 DEG C, and the initial pH of culture medium is 7.2, with ventilation ratio 2.5:1
Carry out fermentation ventilation, mixing speed 200r/min, fermentation time 36h.Final concentration of 8% (v/v) is added in fermentation liquid
Cocoyl esterification compound and final concentration of 1g/L sodium benzoate, pH value is adjusted to 4.5 or so using dilute hydrochloric acid, as
JSPB14 biocontrol agent B, then sterile filling, room temperature preservation.
Application method of the biocontrol agent B in prevention and treatment bacterial leaf streak of rice：Biocontrol agent B is diluted 60~80 times,
Even spraying is carried out in the early period of origination of rice bacterial disease cecospora spot, 7~9d of interval is sprayed 1 time again, can be obtained good anti-
Control effect.
Application case：
In 2017, field control bacterial leaf streak of rice plot experiment is carried out using JSPB14 biocontrol agent B, is investigated
To bacterial leaf streak of rice control efficiency, 4 processing of test setting under its test dose：Clear water control, JSPB14 biocontrol microorganisms
750mL/ mus of agent B, 1000mL/ mus of JSPB14 biocontrol agent B, 100mL/ mus of 20% thiazole zincium suspension agent use water 60kg per acre,
Even spraying.Rice varieties are Y two excellent 900, see disease in investigation in August 13rd (Rice Heading blooming stage).In 15 first times of August
Application, second on the 22nd of August application, 8d and 15d investigates disease index after second of medication.The results are shown in Table 1,
The preventive effect of 8d after second of medication, JSPB14 750mL/ mus of dosage of biocontrol agent B reaches 75.9%, JSPB14 biocontrol agent B
The preventive effect of 1000mL/ mus of dosage reaches 79.1%, (82.6%) close with the preventive effect of 20% thiazole zincium suspension agent of chemical agent；The
15d is investigated after secondary medication, and the preventive effect of JSPB14 750mL/ mus of dosage of biocontrol agent B is declined slightly (72.3%), but
The preventive effect of JSPB14 1000mL/ mus of dosage of biocontrol agent B reaches 80.1%, with 20% thiazole zincium suspension agent preventive effect (79.8%)
It is essentially identical.
1 JSPB14 biocontrol agent B prevention and control bacterial leaf streak of rice field test results of table
Above-described embodiment illustrates that Bacillus amyloliquefaciens strain JSPB14 of the present invention bursts to including adopting rear Pleurotus eryngii
The various plants pathogenetic bacteria such as rotten germ, xanthomonas oryzae pv. oryzicola has stronger antagonistic activity, bursts to rear Pleurotus eryngii is adopted
Rotten disease and bacterial leaf streak of rice have preferable control effect, can reduce or avoid chemical pesticide and farm antibiotics
Residual improves food and Environmental security, has the good prospect of marketing.
It should be noted that above-described embodiment is only presently preferred embodiments of the present invention, there is no for the purpose of limiting the invention
Protection scope, the equivalent substitution or substitution made on the basis of the above all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JSPB14, which is characterized in that strain is protected
Hiding number is CGMCC No.14934.
2. a kind of tunning of bacillus amyloliquefaciens JSPB14 as described in claim 1.
3. one kind is sent out comprising bacillus amyloliquefaciens JSPB14 as described in claim 1 and/or bacillus amyloliquefaciens JSPB14
The biocontrol agent of ferment product.
4. a kind of preparation method of biocontrol agent as claimed in claim 3, which is characterized in that include the following steps：
(1) activation culture：Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, streak inoculation to fresh 2YT
On solid plate, for 24 hours~36h is cultivated under the conditions of 30 DEG C；
(2) prepared by seed liquor：In one single colonie inoculation 2YT fluid nutrient medium of picking, shaken cultivation 16h~36h at 30 DEG C, vibration
Swinging frequency is 100r/min~200r/min；
(3) preparation of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution：Step (2) resulting seed liquor is inoculated into Landy training with the ratio column of 1%~5% (v/v)
It supports and ferments in base, fermentation temperature is 26 DEG C~31 DEG C, and the initial pH of culture medium is 6.5~7.2, with ventilation ratio 0.5:1 arrives
2.5:Fermentation ventilation is carried out between 1, mixing speed is 75r/min~200r/min, and fermentation time is 36h~48h；
(4) preparation of biocontrol agent A：The biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution centrifugation that will be fermented in above-mentioned steps (3), takes sterile supernatant, adds sorb
Sour potassium, then sterile filling, room temperature preservation.
5. preparation method as claimed in claim 4, which is characterized in that in the step (4), add the final concentration of potassium sorbate
For 1g/L.
6. a kind of preparation method of biocontrol agent as claimed in claim 3, which is characterized in that include the following steps：
(1) activation culture：Take the glycerol strain of Bacillus amyloliquefaciens strain JSPB14 of the present invention, streak inoculation to fresh 2YT
On solid plate, for 24 hours~36h is cultivated under the conditions of 30 DEG C；
(2) prepared by seed liquor：In one single colonie inoculation 2YT fluid nutrient medium of picking, shaken cultivation 16h~36h at 30 DEG C, vibration
Swinging frequency is 100r/min~200r/min；
(3) preparation of biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution：Step (2) resulting seed liquor is inoculated into Landy training with the ratio column of 1%~5% (v/v)
It supports and ferments in base, fermentation temperature is 26 DEG C~31 DEG C, and the initial pH of culture medium is 6.5~7.2, with ventilation ratio 0.5:1 arrives
2.5:Fermentation ventilation is carried out between 1, mixing speed is 75r/min~200r/min, and fermentation time is 36h~48h；
(4) preparation of biocontrol agent B：Addition cocoyl esterification compound and benzene in the fermentation liquid prepared in above-mentioned steps (3)
PH value is adjusted to 4.5 or so, then sterile filling, room temperature preservation by sodium formate.
7. preparation method as claimed in claim 6, which is characterized in that cocoyl described in the step (4) is esterified compound
Final concentration of 8% (v/v), the final concentration of 1g/L of sodium benzoate.
8. bacillus amyloliquefaciens JSPB14 or its tunning or the biocontrol agent exist as described in claim any one of 1-7
The application in rear Pleurotus eryngii ulcer disease and bacterial leaf streak of rice is adopted in prevention and treatment.
9. application method of the biocontrol agent A after prevention and treatment is adopted in Pleurotus eryngii ulcer disease, feature exist as described in claim 4 or 5
In, by biocontrol agent A dilute 1~5 times, using immersion way handle adopt rear Pleurotus eryngii, after drying according to adopt rear shelf conditions into
Row is placed.
10. application method of the biocontrol agent B in prevention and treatment bacterial leaf streak of rice, feature exist as claimed in claims 6 or 7
In by 60~80 times of biocontrol agent B dilution, in the early period of origination progress even spraying of rice bacterial disease cecospora spot, 7~9d of interval
It sprays again 1 time.
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| CN113412858A (en) * | 2021-04-28 | 2021-09-21 | 四川大学 | Method for preserving blood orange by using bacillus amyloliquefaciens M73 fermentation product |
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| CN113412858A (en) * | 2021-04-28 | 2021-09-21 | 四川大学 | Method for preserving blood orange by using bacillus amyloliquefaciens M73 fermentation product |
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