CN108384734A - A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease - Google Patents
A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease Download PDFInfo
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- CN108384734A CN108384734A CN201810153593.3A CN201810153593A CN108384734A CN 108384734 A CN108384734 A CN 108384734A CN 201810153593 A CN201810153593 A CN 201810153593A CN 108384734 A CN108384734 A CN 108384734A
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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
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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
Abstract
The invention belongs to technical field of microbe application more particularly to a kind of biocontrol agents and preparation method thereof of prevention tobacco rhizome class disease.It is a kind of prevention tobacco rhizome class disease biocontrol agent, which is characterized in that the main ingredient of the biocontrol agent be by 16,098 two kinds of bacterium mixed fermentives of bacterial strain 16029 and bacterial strain zymotic fluid group at.The biocontrol agent of the present invention can have stronger inhibiting effect, the biocontrol agent that can be used as tobacco rhizome class disease to be used the pathogen of tobacco bacterial wilt, tobacco black shank and the main tubers disease of 3 kinds of black root of tobacco simultaneously.Microbial inoculum of the present invention shows good application prospect as biocontrol agent in prevention tobacco rhizome class disease.
Description
Technical field
The invention belongs to technical field of microbe application more particularly to a kind of biocontrol agents of prevention tobacco rhizome class disease
And preparation method thereof.
Background technology
Tobacco is a kind of important leaf with industrial crops, is the main economic source of cigarette district peasant programme, but cigarette
Grass growth is seriously affected by tubers disease bacterial wilt, balck shank and root black rot, tobacco rhizome class disease the torrid zone and
Subtropical zone cigarette district is particularly acute, and is the destructive disease for threatening World tobacco production.3 kinds of main tubers diseases are often mixed
Morbidity is closed, the extent of injury is aggravated, the old sick field in many grave illness areas or nonirrigated farmland vega, full field are fallen ill and had no harvest without annual the phenomenon that receipts
All occur, cause yield of tobacco and quality decline and caused by directly quality loss it is very serious.Tobacco bacterial wilt is by Solanaceae
A kind of bacillary vascular bundle diseases caused by La Er Salmonella Ralstonia solanacearum.Disease Main Tobacco Planting in south China
The provinces such as area such as Guangxi, Guangdong, Fujian, Hunan, Zhejiang (autonomous region) generally occur, and onset area is larger, cause harm serious, in recent years
Also north cigarette district extension trend, such as Shandong, Henan, Shaanxi and Liaoning province all oneself have the distribution of the disease and cause to cause harm
Record, local cigarette district remains serious.Tobacco black shank is one of most destructive disease, also known as tobacco on tobacco leaf production
Epidemic disease, each main production cigarette district of China have different degrees of generation, and wherein Anhui, Shandong, Henan Province are historical grave illness area, should
Disease often mixes generation with tobacco bacterial wilt, therefore endangers even more serious.Since continuous cropping vega area expands in recent years, this disease causes harm and has
The gesture of apparent rising.Tobacco black shank cause of disease is phytophthora parasitica tobacco pathogen type (Phytophtora parasitica
Var.nicotianae Tucker), it is Oomycete fungal.The main cane of causing harm of the disease, disease plant basal part of stem are in just water stain shape
Blackspot, it is backward up and down and marrow extension, when around stem one week, plant is wilted death, vertical profile disease stem, it is seen that the necrosis of marrow dark brown is simultaneously
In laminated, white flock mycelia is covered with.Black root of tobacco is also global disease, Yunnan Province of China, Guizhou, Hubei,
The generation of the provinces and regions such as Guangxi is heavier, and serious plot incidence also has the tendency that aggravating harm in recent years up to 30% or more.Tobacco Root is black
Maize ear rot cause of disease thielaviopsis basicola be base take root string strain it is mould, belong to Deuteromycotina fungi.The disease the seedbed phase with
And when field planting can occurring and damage, Seedling Stage morbidity, germ invades from the native exterior portion position of seedling first, and scab is around stem
Portion, and cotyledon is invaded upwards, cause to rot, destroys tobacco seedlings；Field period is fallen ill, and cigarette strain initial stage aerial part shows growth retardation,
It is easy to wilt, visible tip of a root browning of uprooting, dark brown to rot, all at special black, fibrous root is often difficult to pull out severe patient's old complaint
Go out, residual is in the soil.
The 3 main source of infection for growing tobacco tubers disease derive from soil, and field of especially falling ill over the years is deposited in prevention
In bigger difficulty.Especially tobacco bacterial wilt, since the disease belongs to bacterial disease, the susceptible category systematicness of plant is disseminated, because
And it prevents very difficult；Tobacco black shank and root black rot belong to fungal disease, though there are some opposite in chemical prevention at present
Relatively effective medicament, but cause harm that there is also bigger difficulty to radical cure pathogen, crop field plant is once susceptible, and the state of an illness is difficult to
Control.In addition, chemical agent to the prevention of tobacco rhizome class disease there is also many shortcomings, germ easily produces chemical pesticide
Raw drug resistance, largely can cause environmental pollution and the rising of production cost using chemical pesticide.In recent years, research is more is
Biological control and induced resistance.Biological control have many advantages, such as it is nuisanceless, pollution-free, be prevention tobacco rhizome class disease on
It is a kind of economical, effectively and sustainable approach to development.Using biocontrol microorganisms prevention bacterial wilt be made in recent years more research it
One, such as the wide Pseudomonasspp. with antagonism of sieve obtains favorable effect in greenhouse prevention tobacco bacterial wilt, but is not applied to
Crop field；The Anuratha and Gnanamanick of India carry out the efficiency test of various crop bacterial wilt with P.fluorescens,
Obtain good preventive effect.Hayward etc. has found that VA Mycorrhizal Fungi can mitigate the generation of bacterial wilt, Suresh etc. in Filipine research
It was found that bubble assists arbuscular mycorrhiza extract to have strong bacteriostasis to the growth and breeding of Ralstonia solanacearum.Biological control method can be at present
Mitigate bacterial wilt to a certain extent to cause harm, but there is also the limitations such as preventive effect is unstable in current practice.Due to field
By there is a phenomenon where a variety of tubers diseases to mix occurring and damage, therefore, 3 big tubers diseases can be inhibited simultaneously by how screening
Biocontrol agent and its optimal use method are urgent problems to be solved in tobacco rhizome class disease control.
Burkholderia (Burkholderia cepacia) is that one kind being widely present in water, soil, plant
With the gram-Negative bacillus in human body, become Major Members in biological control because it can generate a variety of antimicrobial components.
Recent studies indicate that not instead of one kind of Burkholderia, one group phenotype is close but genotype is different
Compound, referred to as Burkholderia group (Burkholderia cepacia complex, abbreviation BCC), so far
It was found that BCC includes 20 different genotype, due to there is very low DNA hybridization rate between these genotype, can represent not
Same kind.At the beginning of the last century 80's, people have found that some in the degradable pesticide of part BCC bacterial strains and herbicide are grown under study for action
There is and have to people the chemical substance of potential carcinogenesis in the phase, can also purify sewage, have extraordinary ecology and economic value；
Simultaneously it has also been found that some BCC bacterial strains have the function of preventing plant pest and promote plant growth, it is made into biological insecticides
And biological bactericide, it plays an important role in agricultural production.But at the same time people have found some bacterial strains of the bacterium again
It is opportunistic human bacterial pathogen.Some American-European countries abandon or limit BCC biology agricultures because its part bacterial strain is conditioned pathogen
The use of medicine, but still have many countries in production and use using it as biological pesticide made of raw material at present, China is also in 1996
Year has registered such product.
BCC is made of 20 kinds (or genotype), including B.cepacia, B.multivorans, B.cenocepacia,
B.stabilis、B.vietnamiensis、B.dolosa、B.ambifaria、B.anthina、B.pyrrocinia、
B.ubomensis、B.latens、B.diffusa、B.arboris、B.seminalis、B.metallica、
B.contaminans, B.lata, B.pseudomultivorans, B.stagnalis and B.territorii.These kinds are certainly
Right boundary generally existing, it is especially close with plant relations.At present the study found that BCC pathogenic strains and specific virulence gene
(BCESM genes) there are correlations.BCESM genes are considered as a kind of in the BCC bacterium and chain heredity of human body cause illness's property/toxicity
Label, to chartered Burkholderia strain, once find the presence of such label, then un-register.Therefore,
By the detection of virulence factor BCESM genes, the pathogenic strain and non pathogenic strain of BCC can be distinguished.Certainly, in order to ensure
The safety that BCC biocontrol bacterial strains use further detects whether bacterial strain has using animal model (small white mouse) or plant model
It is pathogenic to be also necessary.Clover is generally used for the model plant of hospital's Bacteria infecting respiratory Pathogenicity.People study
It was found that clover can replace the toxicity that mouse measures BCC floras to mammal as model plant, clover model even to show
Sensitivity more higher than mouse models is gone out.Therefore, it can quickly and easily detect BCC bacterial strains to mammal with clover model
Whether have pathogenic, lays a good foundation for safe utilization BCC bacterial strains.
Being disclosed in the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without answering
It has been the prior art well known to persons skilled in the art when being considered as recognizing or imply that the information is constituted in any form.
Invention content
It is an object of the present invention to provide a kind of biocontrol agent and preparation method thereof, it is withered which can effectively control tobacco blueness
3 kinds of disease, balck shank and root black rot diseases, and the biocontrol agent remarkably promotes effect to tobacco growing.
Technical solution provided by the invention is as follows：
A kind of biocontrol agent of prevention tobacco rhizome class disease, the active constituent of the biocontrol agent is by bacterial strain
16029 and 16,098 two kinds of bacterium mixed fermentives of bacterial strain zymotic fluid group at.
Preferably, the taxology of bacterial strain 16029 was named as Burkholderia anthina, on 01 10th, 2018
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC NO.15165.
Preferably, the taxology of bacterial strain 16098 was named as Burkholderia cenocepacia, in 01 month 2018
It is preserved within 10th China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC NO.15166.
The present invention also provides the preparation methods of the biocontrol agent of the prevention tobacco rhizome class disease, including walk as follows
Suddenly：
(1) prepared by seed liquor：Bacterial strain 16029 and bacterial strain 16098 are seeded in respectively on nutrient culture medium NA independent
Culture, 30 DEG C of shaking table shaken cultivation 20-24h, by two strain cultured solution mixed in equal amounts to get the seed liquor of biocontrol agent；
(2) acquisition of bacteria fermentation liquid：The seed liquor of cultured biocontrol agent is taken to be inoculated on fermentation medium, 25-
28 DEG C of shaken cultivation 30-36h, obtain the zymotic fluid of biocontrol agent；
(3) acquisition of biocontrol agent of the present invention：Microbial inoculum additive is added into the zymotic fluid of biocontrol agent, is stirred evenly,
Up to biocontrol agent.
Preferably, fermentation medium is in step (2)：Fructus hordei germinatus leaching powder 2.0g/L, sucrose 8.0g/L, yeast powder 2g/L,
Potassium dihydrogen phosphate 0.5g/L, manganese sulfate 0.3g/L, NaCl 3.0g/L, pH value 6.5-7.0.
Preferably, the inoculum concentration of seed liquor is 3% in step (2).
Preferably, microbial inoculum additive is by wheat bran and beancake powder in mass ratio 2 in step (3):1 mixing, drying and powder
It is obtained after broken.
Preferably, the additive amount of microbial inoculum additive is 2% in step (3).
Compared with prior art, the present invention has the advantages that：
(1) biocontrol agent of the invention can be main to 3 kinds of tobacco bacterial wilt, tobacco black shank and black root of tobacco simultaneously
There is the pathogen of tubers disease stronger inhibiting effect, the biocontrol agent that can be used as tobacco rhizome class disease to be used.
Microbial inoculum of the present invention shows good application prospect, potting controlling experiment as biocontrol agent in prevention tobacco rhizome class disease
The result shows that inoculation microbial inoculum to the control effect of tobacco bacterial wilt up to 84.77%；And daejeon prevention test is to tobacco bacterial wilt
Control effect also reaches 80.64%, reaches 85% or more to the control effect of balck shank and root black rot, being significantly better than
The control effect for learning medicament, preferably controls causing harm for tobacco rhizome class disease.
(2) biocontrol agent of the invention has Soluble phosphorus and potassium decomposing characteristic simultaneously, is inoculated with the biocontrol agent and has to tobacco growing
Remarkably promote effect.
(3) biocontrol agent of the invention is easy culture, and fermentation condition is simple, easy to operate, as long as providing well-grown
Seed liquor, general tobacco grower can be enlarged using easy fermentation condition culture obtain bacteria fermentation liquid, the fermentation after culture
It may be directly applied to produce after liquid addition biological and ecological methods to prevent plant disease, pests, and erosion auxiliary agent, there is good popularizing application prospect.
(4) use of fertilizer and pesticide, preserving the ecological environment is of great significance biocontrol agent of the invention for reducing.
Description of the drawings
Fig. 1 is the fungistatic effect of two plants of BCC bacterial strains；
Fig. 2 is growth-promoting effect of the biocontrol agent to tobacco of the present invention；
Fig. 3 is that the biocontrol agent of the present invention prevents the pot experiment effect of tobacco bacterial wilt；
Fig. 4 is BCC bacterial strains used in the biocontrol agent of the present invention to the pathogenic testing result of tobacco；
Fig. 5 is BCC bacterial strains used in the biocontrol agent of the present invention to the pathogenic testing result of clover.
Preservation information explanation
Burkholderia anthina, deposit number are CGMCC NO.15165, and the deposit date is 01 month 2018
10, depositary institution was China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of city Chaoyang District North Star West Road 1, postcode：100101)；
Burkholderia cenocepacia, deposit number are CGMCC NO.15166, and the deposit date is 2018
10 days 01 month, depositary institution was 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, postcode：100101).
Specific implementation mode
Following embodiment does not limit the present invention to be better understood from the present invention.Experimental method in following embodiments,
Unless otherwise specified, it is conventional method.Experiment material as used in the following examples is unless otherwise specified conventional life
Change what reagent shop was commercially available.
Quantitative experiment in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Embodiment 1：The screening and identification of bacterial strain
(1) screening of bacterial strain
(bacterial strain original number is respectively 16029 Hes to 2 plants of biocontrol microorganisms that complex micro organism fungicide of the present invention is included
16098) it is that separation screening comes out from the cassava root soil of Guangxi Zhuang Autonomous Region Beihai Hepu County cassava main producing region
, above-mentioned bacterial strains were stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 01 month 2018, letter
The deposit number of title CGMCC, bacterial strain 16029 and bacterial strain 16098 is respectively CGMCC No.15165 and CGMCC No.15166.
(2) taxonomic identification of bacterial strain
By colonial morphology, 16S rDNA sequence analyses, Biolog Bacteria Identifications system, BCC floras specific amplification
The analyses such as the different genotype identification primer detection of PCR, BCC, are CGMCC No.15165 and CGMCC by deposit number
Two plants of bacteriums of No.15166 are identified respectively as garden bulkholderia cepasea (Burkholderia anthina) and new onion
Bulkholderia cepasea (Burkholderia cenocepacia).
Embodiment 2：The preparation of biocontrol agent
The zymotic fluid of biocontrol agent of the present invention by bacterial strain 16029 and 16098 seed liquor of bacterial strain mixed culture obtain, based on
Lower experiment screening result：
(1) bacterial strain 16029 and bacterial strain 16098 all have a Soluble phosphorus potassium decomposing effect, but the Soluble phosphorus potassium decomposing effect of bacterial strain 16029
It is more better than bacterial strain 16098.
(2) two bacterial strains can inhibit the important phytopathogen such as tobacco bacterial wilt, have Biocontrol Potential, but bacterial strain 16098
Biocontrol effect it is much better compared with 16029.The bacteria suspension and culture stoste of two bacterial strains have stronger bacteriostasis, but sterile mistake
The equal unobvious of filtrate fungistatic effect.Fig. 1 is shown in the fungistatic effect screening of two plants of BCC bacterium.
(3) under condition of culture unanimous circumstances, the growth curve of two bacterial strains is almost the same.
(4) by measuring the influence of different carbon source, two strain growth of nitrogen source and inorganic salts pair, the seed culture of microbial inoculum is determined
Base is ordinary nutritional bouillon media NA (beef extract 3.0g, peptone 10.0g, NaCl5.0g add and be settled to 1L, pH7.0)；Bacterium
Agent expand culture (fermentation) culture medium be：Fructus hordei germinatus leaching powder 2.0g/L, sucrose 8.0g/L, yeast powder 2g/L, potassium dihydrogen phosphate
0.5g/L, manganese sulfate 0.3g/L, NaCl 3.0g/L, pH value 6.5-7.0.
The preparation method of the biocontrol agent of the prevention tobacco rhizome class disease, includes the following steps：
(1) acquisition of microbial inoculum seed liquor of the present invention：Bacterial strain 16029 and bacterial strain 16098 are seeded in respectively on NA culture mediums
Two strain cultured solution mixed in equal amounts are the seed liquor of microbial inoculum of the present invention by independent culture, 30 DEG C of shaking table shaken cultivation 20-24h.
(2) acquisition of bacteria fermentation liquid：Cultured seed liquor is taken to be inoculated on fermentation medium, inoculum concentration 3%,
25-28 DEG C of shaken cultivation 30-36h up to microbial inoculum of the present invention zymotic fluid.
(3) prepared by the additive (auxiliary agent) of microbial inoculum of the present invention：Wheat bran and beancake powder press 2:1 (weight ratio) mixing drying and powder
It is broken, it is sealed after sterilizing.
(4) acquisition and its use of biocontrol agent of the present invention：2% above-mentioned microbial inoculum additive is added into bacteria fermentation liquid
(auxiliary agent) is biocontrol agent of the present invention, can directly be used in production after diluting corresponding multiple on demand.
Bacteria fermentation culture medium of the present invention is easy to prepare, and fermentation condition is simple, to meet the larger requirement of field usage amount,
General tobacco grower oneself can prepare fermentation medium under conditions of obtaining seed liquor and microbial inoculum helps and carry out simple fermented and cultured,
Zymotic fluid after culture is mixed with auxiliary agent in proportion can be used directly.
Embodiment 3：The Plant growth-promoting effect characteristic of the biocontrol agent of the present invention
(1) soil incubation method measures the Soluble phosphorus potassium decomposing effect of microbial inoculum
It is mixed in equal volume with seedling medium with cultivating reddle loam substantially, and appropriate calcium phosphate and feldspar in powder is added, mixed
Uniform rear bottling sterilizing is closed, is inoculated with bacteria fermentation liquid of the present invention, inoculum concentration 3% compares and connects equivalent aseptic culture fluid, after 20d
Measure the available phosphorus of each processing and effective potassium content.Available phosphorus is measured using molybdenum antimony resistance colorimetric method, and effective potassium then uses flame light
Degree method measures.
The experimental results showed that inoculation processing early period mixed-matrix available phosphorus and effective potassium content be respectively 8.2mg/kg and
117.6mg/kg；It is inoculated with the available phosphorus of microbial inoculum processing after 20d and effective potassium content distinguishes 14.2mg/kg and 144.5mg/kg, and
Control treatment is respectively then 8.6mg/kg and 119.0mg/kg.
As it can be seen that the available phosphorus and effective potassium content of matrix have a more substantial increase after inoculation biocontrol agent processing 20d, and
Respectively 65.12% and 21.43% are improved than control treatment.
(2) growth promoting function of biocontrol agent of the invention to tobacco seedling
Tobacco seedling basin is handled through conventional seedbed system, and tobacco seedlings are sprayed after growing two panels true leaf with 30 times of dilutions of microbial inoculum of the present invention
2 times (interval 7d), control sprays equivalent clear water.
As shown in Fig. 2, the processed tobacco seedlings growing way of biocontrol agent of the present invention is substantially better than control, microbial inoculum inoculation processing
The leaf area of tobacco seedling blade, chlorophyll content improve 27.5% and 34.8% than control respectively.
Embodiment 4：Controlling experiment of the biocontrol agent of the present invention in prevention tobacco rhizome class disease
(1) the potting inoculation test of microbial inoculum prevention tobacco bacterial wilt of the present invention
Cultivation soil in pot experiment is the topsoil of healthy rice terrace, tobacco routinely culturing and transplanting seedlings, per basin list
Strain is planted, and is inoculated with 30 times of dilution zymotic fluids of microbial inoculum of the present invention after transplanting 7d, 14d and 21d respectively, and each single plant is inoculated with 200mL,
Control connects equivalent clear water.The inoculation of tobacco ralstonia solanacearum then selects this research team from the bacterial wilt of Baise of Guangxi tobacco main producing region
Separation screening obtains the mixed bacteria liquid with 3 plants of Lao Ershi Ralstonia solanacearums compared with High pathogenicity, inoculum density 10 in strain8cfu/
ML, the inoculation bacteria suspension 150mL per basin (strain) pouring root；The first time inoculation time of tobacco ralstonia solanacearum is 10d after transplanting, after
It was inoculated with equivalent cause of disease bacterium solution 1 time every 3 days, apparent bacterial wilt symptom occurs in 5-8 extremely control of vaccinization.(CK) is compareed to start
Start to carry out continuous observation to the incidence of tobacco after there is typical bacterial wilt symptom, disease plant number is recorded, according to following
Formula calculates the opposite control effect of microbial inoculum inoculation：
Preventive effect={ (blank control handles diseased plant rate-Prevention technology area diseased plant rate)/blank control handles diseased plant rate } ×
100。
Experimental study shows that control bacterial wilt symptom occurs to tobacco transplant 25d (Ralstonia solanacearum is inoculated with 15d) afterwards, connects
Then just there are individual plant disease symptoms for the first time after transplanting 30d in the tobacco seedlings of kind biocontrol agent processing of the present invention；After transplanting 50d
The tobacco diseased plant rate for being inoculated with microbial inoculum processing is 11.5%, and all 1 grade of diseased plants, and the diseased plant rate compareed is 75.5%, and
The 30% diseased plant state of an illness reaches 7-9 grades.Microbial inoculum of the present invention is calculated according to diseased plant rate to reach the control effect of tobacco bacterial wilt
84.77%.
(2) field experiment of the biocontrol agent of the invention prevention main tubers disease of 3 kinds of tobacco
Field plot controlling experiment sets 3 processing, microbial inoculum processing, Agro-chemicals control processing and blank respectively of the present invention
It compares (CK does not do Prevention technology), processing scheme refers to table 1.Often processing sets 4 replicated plots, is about 0.08 per plot area
Mu, 80 plants of tobacco strain number, i.e. the total experiment tobacco strain number of often processing is 320 plants.
1 tobacco rhizome class disease cell controlling experiment processing scheme of table
Tobacco bacterial wilt, the investigation of tobacco black shank and black root of tobacco and grade scale are pressed《Tobacco diseases are classified
And investigation method》(China tobacco business standard YC/T 39-1996).
Disease index and control effect are calculated as follows：
Disease index={ ∑ (the diseased plant numbers at different levels × disease grade value)/(investigation total strain number × superlative degree value) } × 100
Opposite control effect (%)={ (control disease index-processing disease index)/control disease index } × 100
The disease index and control effect (being shown in Table 2) of each processing are calculated according to last time investigation result.
2 tobacco of table, 3 kinds of tubers disease plot experiment control effects
As shown in table 2, biocontrol agent of the invention reaches 80.64% to the control effect of tobacco bacterial wilt, to balck shank
And the control effect of root black rot then respectively reaches 85.13% and 87.83%, control effect is substantially better than chemical agent,
Causing harm for 3 kinds of diseases of tobacco can effectively be controlled.
Embodiment 5：The safety evaluatio of the biocontrol agent of the present invention
The BCC bacterial strains used in biocontrol agent are determined respectively to onion, tobacco leaf and clover whether with pathogenic；Together
When, by PCR specific amplifications, detection bacterial strain whether the virulence factor (BCESM) containing BCC floras, to prove microbial inoculum pair
Whether plant, people and animals and ecological environment are safe.
(1) to the pathogenic detection of onion and tobacco leaf
Different BCC bacterial strains distinguish activation culture, are inhaled to the pathogenic detection 1ml primary sterilization syringes of onion bulb stem
100 μ l are taken to contain 107The bacteria suspension of the strains tested of a cell, the fresh and healthy onion bulb stem of stab inoculation, is made with sterile water
Control.In being cultivated under conditions of 30 DEG C, relative humidity 95% and 12h light/12h dark in growth cabinet after inoculation, see daily
Examine whether onion bulb stem rots.With the bacterial suspension inoculation tobacco of same concentration, with disposable inoculation syringe 1ml bacteria suspension pressures
Enter blade, to inject 1ml sterile waters as a contrast.After 5-7d, whether observation injection site will appear brown hypersensitive necrosis spot.
As shown in figure 4, after inoculation onion bulb stem 7d, the not rotten sign of onion bulb stem；Tobacco leaf inoculation after, equally with
Control is no different, and hypersensitive necrosis spot does not occur.This research shows that all two plants of BCC bacterium of microbial inoculum of the present invention to onion and tobacco
Without pathogenic, bacterial strain is not phytopathogen, to plant safety.
(2) pathogenic detection of the strains tested to clover
Clover is to replace mouse as measurement BCC to the model plant of animal toxicity, can be quickly and easily with clover model
It is pathogenic to detect whether BCC bacterial strains have mammal.
It will be respectively inoculated into LB culture mediums for two bacterial strains of examination, for 24 hours, centrifugation is received for 30 DEG C of shaking table culture (180rpm) cultures
Collect thalline, with sterile water tune bacteria suspension concentration to 108/ mL is for being inoculated with.Alfalfa seed is impregnated with 98% concentrated sulfuric acid
Then 20min is steeped in sterile water with sterile water wash 4 times, 30 DEG C of seed soaking 7h, then is cleaned 2 times, impregnates 10h again after changing water,
Then seed is placed on water agar plate and prepares to be inoculated with.Seed on agar plate transfer needle is pierced into an aperture, takes 20 μ
The ready bacteria suspension points of L are seeded in wound, as a contrast with the seed of stab inoculation sterile water.Seed after inoculation is placed in
It is cultivated in 28 DEG C of illumination boxs, inoculation incidence is checked after 7d.
As shown in figure 5, the processing of inoculation strains tested is compareed with sterile water equally without disease symptom, vaccination attachment without
, there is not total aetiolation in necrosis phenomena, seminal root and the equal well-grown of cotyledon.It should be the result shows that strains tested be dynamic to lactation
Object is safe.
(3) strains tested whether the detection containing BCC virulence factors BCESM
The detection of virulence factor B.cepacia epidemic strain marker (BCESM) sequence of BCC floras is used
The primer BCESM1 of Mahenthiralingam (1997) designs：5'-CCACGGACGTGACTAACA-3' and BCESM2：5'-
CGTCCATCCGAACACGAT-3' carries out PCR amplification, and amplified production is detected with 1.5% agarose gel electrophoresis, uses gel imaging
Analyzer is imaged.
It is detected through PCR, two plants of strains testeds do not amplify the BCESM virulence factor sequences of about 1400bp.
The description of the aforementioned specific exemplary embodiment to the present invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining the specific principle of the present invention and its actually answering
With so that those skilled in the art can realize and utilize the present invention a variety of different exemplary implementation schemes and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Sequence table
<110>Institute of Microbiology, Guangxi Academy of Agricultural Sciences
<120>A kind of biocontrol agent and preparation method thereof of prevention tobacco rhizome class disease
<130> ZYWS
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1394
<212> DNA
<213> Burkholderia sp.
<400> 1
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acatcggaac atgtcctgta gtgggggata gcccggcgaa agccggatta ataccgcata 120
cgatctacgg atgaaagcgg gggaccttcg ggcctcgcgc tatagggttg gccgatggct 180
gattagctag ttggtggggt aaaggcctac caaggcgacg atcagtagct ggtctgagag 240
gacgaccagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaattttgga caatgggcga aagcctgatc cagcaatgcc gcgtgtgtga agaaggcctt 360
cgggttgtaa agcacttttg tccggaaaga aatccttggc tctaatacag ccgggggatg 420
acggtaccgg aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg 480
gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg caggcggttt gctaagaccg 540
atgtgaaatc cccgggctca acctgggaac tgcattggtg actggcaggc tagagtatgg 600
cagagggggg tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg 660
atggcgaagg cagccccctg ggccaatact gacgctcatg cacgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc ctaaacgatg tcaactagtt gttggggatt 780
catttcctta gtaacgtagc taacgcgtga agttgaccgc ctggggagta cggtcgcaag 840
attaaaactc aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggattaattc 900
gatgcaacgc gaaaaacctt acctaccctt gacatggtcg gaatcctgct gagaggcggg 960
agtgctcgaa agagaaccgg cgcacaggtg ctgcatggct gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgtcc ttagttgcta cgcaagagca 1080
ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttatgg gtagggcttc acacgtcata caatggtcgg aacagagggt tgccaacccg 1200
cgagggggag ctaatcccag aaaaccgatc gtagtccgga ttgcactctg caactcgagt 1260
gcatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gtcttgtaca caccgcccgt cacaccatgg gagtgggttt taccagaagt ggctagtcta 1380
accgcaagga ggac 1394
<210> 2
<211> 1393
<212> DNA
<213> Burkholderia sp.
<400> 2
gcagtcgaac ggcagcacgg gtgcttgcac ctggtggcga gtggcgaacg ggtgagtaat 60
acatcggaac atgtcctgta gtgggggata gcccggcgaa agccggatta ataccgcata 120
cgatctatgg atgaaagcgg gggaccttcg ggcctcgcgc tatagggttg gccgatggct 180
gattagctag ttggtggggt aaaggcctac caaggcgacg atcagtagct ggtctgagag 240
gacgaccagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaattttgga caatgggcga aagcctgatc cagcaatgcc gcgtgtgtga agaaggcctt 360
cgggttgtaa agcacttttg tccggaaaga aatccttggc tctaatacag tcgggggatg 420
acggtaccgg aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg 480
gtgcgagcgt taatcggaat tactgggcgt aaagcgtgcg caggcggttt gctaagaccg 540
atgtgaaatc cccgggctca acctgggaac tgcattggtg actggcaggc tagagtatgg 600
cagagggggg tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg 660
atggcgaagg cagccccctg ggccaatact gacgctcatg cacgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc ctaaacgatg tcaactagtt gttggggatt 780
catttcctta gtaacgtagc taacgcgtga agttgaccgc ctggggagta cggtcgcaag 840
attaaaactc aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggattaattc 900
gatgcaacgc gaaaaacctt acctaccctt gacatggtcg gaatcctgct gagaggcggg 960
agtgctcgaa agagaaccgg cgcacaggtg ctgcatggct gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgtcc ttagttgcta cgcaagagca 1080
ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttatgg gtagggcttc acacgtcata caatggtcgg aacagagggt tgccaacccg 1200
cgagggggag ctaatcccag aaaaccgatc gtagtccgga ttgcactctg caactcgagt 1260
gcatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gtcttgtaca caccgcccgt cacaccatgg gagtgggttt taccagaagt ggctagtcta 1380
accgcaagga gga 1393
Claims (8)
1. a kind of biocontrol agent of prevention tobacco rhizome class disease, which is characterized in that the active constituent of the biocontrol agent is
By 16,098 two kinds of bacterium mixed fermentives of bacterial strain 16029 and bacterial strain zymotic fluid group at.
2. the biocontrol agent of prevention tobacco rhizome class disease according to claim 1, which is characterized in that bacterial strain 16029
Taxology is named as Burkholderia anthina, and Chinese microorganism strain preservation management was preserved on 01 10th, 2018
Committee's common micro-organisms center, preserving number are CGMCC NO.15165.
3. the biocontrol agent of prevention tobacco rhizome class disease according to claim 1, which is characterized in that bacterial strain 16098
Taxology is named as Burkholderia cenocepacia, and Chinese microorganism strain preservation was preserved on 01 10th, 2018
Administration committee's common micro-organisms center, preserving number are CGMCC NO.15166.
4. the preparation method of the biocontrol agent of prevention tobacco rhizome class disease according to claim 1, which is characterized in that packet
Include following steps：
(1) prepared by seed liquor：Bacterial strain 16029 and bacterial strain 16098 are seeded on nutrient culture medium NA independent culture respectively,
30 DEG C of shaking table shaken cultivation 20-24h, by two strain cultured solution mixed in equal amounts to get the seed liquor of biocontrol agent；
(2) acquisition of bacteria fermentation liquid：The seed liquor of cultured biocontrol agent is taken to be inoculated on fermentation medium, 25-28 DEG C
Shaken cultivation 30-36h obtains the zymotic fluid of biocontrol agent；
(3) acquisition of biocontrol agent of the present invention：Into the zymotic fluid of biocontrol agent add microbial inoculum additive, stir evenly to get
Biocontrol agent.
5. the preparation method of the biocontrol agent of prevention tobacco rhizome class disease according to claim 4, which is characterized in that step
Suddenly fermentation medium is in (2)：Fructus hordei germinatus leaching powder 2.0g/L, sucrose 8.0g/L, yeast powder 2g/L, potassium dihydrogen phosphate 0.5g/L, sulphur
Sour manganese 0.3g/L, NaCl 3.0g/L, pH value 6.5-7.0.
6. the preparation method of the biocontrol agent of prevention tobacco rhizome class disease according to claim 4, which is characterized in that step
Suddenly the inoculum concentration of seed liquor is 3% in (2).
7. the preparation method of the biocontrol agent of prevention tobacco rhizome class disease according to claim 4, which is characterized in that step
Suddenly microbial inoculum additive is by wheat bran and beancake powder in mass ratio 2 in (3):1 mixing is dried and is obtained after crushing.
8. the preparation method of the biocontrol agent of prevention tobacco rhizome class disease according to claim 4, which is characterized in that step
Suddenly the additive amount of microbial inoculum additive is 2% in (3).
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| CN114214259A (en) * | 2022-01-17 | 2022-03-22 | 中国农业科学院烟草研究所 | Intelligent biocontrol microbial inoculum and preparation method and application thereof |
| CN117187077A (en) * | 2023-07-24 | 2023-12-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
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| CN117187077A (en) * | 2023-07-24 | 2023-12-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
| CN117187077B (en) * | 2023-07-24 | 2024-03-08 | 广东省科学院南繁种业研究所 | Composite flora and application thereof, and control method of bacterial wilt of plants |
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| CN108384734B (en) | 2021-04-30 |
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Effective date of registration: 20210618 Address after: 530007, 174 East University Road, the Guangxi Zhuang Autonomous Region, Nanning Patentee after: GUANGXI ACADEMY OF AGRICULTURAL SCIENCES Address before: No. 174, Daxue East Road, XiXiangTang District, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: INSTITUTE OF MICROBIOLOGY, GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES |