CN109112163A - One plant of Lactobacillus pentosus fermentation liquid and its application for inhibiting phytophthora blight of pepper - Google Patents
One plant of Lactobacillus pentosus fermentation liquid and its application for inhibiting phytophthora blight of pepper Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to biological control soil-borne pathogen technical field, one plant of Lactobacillus pentosus fermentation liquid is provided and its inhibits the application of phytophthora blight of pepper.Lactobacillus pentosus (Lactobacillus Pentosus) China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on January 16th, 2007, deposit number is CGMCC No.1919.Crop, vegetables are avoided using the residual of chemical pesticide caused by chemical pesticide, ensure that the edible safety of crop, vegetables.The accumulation of chemical pesticide in the soil is alleviated, pollution of the chemical pesticide to soil is prevented.Pollution will not be generated in the soil.Enable the tellurian mankind and biology normal growth and multiplies from generation to generation.Inhibiting rate can reach 87%, and Lactobacillus pentosus fermented supernatant fluid significantly inhibits phytophthora blight of pepper, can be used in the biological agent of preparation prevention and treatment phytophthora blight of pepper.
Description
Technical field
The invention belongs to biological control soil-borne pathogen technical fields, and in particular to one plant of Lactobacillus pentosus fermentation liquid and its
Inhibit the application of phytophthora blight of pepper.
Background technique
Soil-borne disease is that pest and disease damage breeds in the soil, survives or overwintering, encroaches on ground by communication media of soil
Plant, damaging range is wide, and spread speed is fast, and concealment is strong, therefore loss is huge, and refractory reason.Due to soil-borne disease insect pest
Harm and caused by crop yield loss would generally reach 20%~40%, serious plot may be up to 60%, or even absolutely
It receives.Soil-borne disease seriously constrains the development of protecting field.
With the adjustment of the structure of agricultural production, vegetable cultivation area in all parts of the country expands year by year, the hair of the various soil-borne diseases of vegetables
It changes to get worse.Phytophthora blight of pepper is to endanger a kind of extensive soil-borne pathogen, at present different continents it has been found that
And host range constantly expands, and can endanger 78 kinds of plants of 18 sections at present, threaten the mass production of various plants.Capsicum epidemic disease
Mould can generate egg spore, due to egg spore have resistance, extreme environmental condition can be restrained oneself, phytophthora blight of pepper becomes
It is most difficult to one of the disease of prevention and treatment.There is important realistic meaning to the research of the prevention and treatment of phytophthora blight of pepper.
The application of soil sterilants has good effect to soil-borne pathogen-phytophthora blight of pepper.Especially high-intensitive chemistry
The application of medicament soil disinfection technology, drastically reduce the harm of phytophthora blight of pepper and caused by crop yield lose.But
It is largely to cause crop edible portion pesticide excess residual using chemical pesticide, and easily cause soil hardening, soil fertility decline
And the problems such as environmental pollution.Since its duration of efficacy is short when being prevented and treated at present using chemical bactericide, thus peasant uses medicine
The number and dosage of agent are stepped up, and accumulation of the oxious component in crop, vegetables, soil also gradually increases.This feelings
Condition is especially prominent in vegetables in greenhouse booth cultivation, constitutes high risks to human health and ecological environment.Therefore, drug effect is developed
Persistently, and to human body and ecological environment, harmless bacteria agent substitution chemical pesticide becomes China or even worldwide development
Direction.Currently, largely the microorganism with Biocontrol Potential is studied reports.It, can be from basic by screening antagonistic microbe
The upper growth and breeding for inhibiting phytophthora blight of pepper, to contain capsicum epidemic disease.Importantly, many biological control agents are not only to disease
Opportunistic pathogen shows Antagonism, and can form a kind of beneficial distinctive micro-ecological environment [ painting fine jade etc., northwest in plant rhizosphere
Agriculture and forestry science and technology university, 2004,35(3): 236-245 ], promote absorption and utilization of the plant to extraneous nutriment, or even generate
It is possible that promoting the metabolin of the growth and breeding of plant, this is not only a kind of safe and efficient control method, and is also green
Required for Agro-ecology sustainable development.
Summary of the invention
The object of the present invention is to provide the applications of one plant of Lactobacillus pentosus fermentation liquid and its inhibition phytophthora blight of pepper.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) be preserved in on January 16th, 2007
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.1919.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) it is (to be taken from from Pickles, Sichuan Style long-used soup
Certain Sichuan cuisine wineshop at China Agricultural University east school district east gate, wherein NaCl concentration be 4.75%, pH be 4.19) in isolated, generation
Number be sp, specific separation method are as follows: using dilution 108Spread plate method, (it is separated using MRS fluid nutrient medium, in
Cultivated in 37 DEG C of incubators, culture medium MRS specific formula is as follows) by continuously setting-out flat band method purifying, using API50CH
Reagent strip and API50CHL culture medium (Bio-Merieux, 50410) carry out isolated bacterial strain according to the instruction explanation of manufacturer sugared
Fermentation reaction, test result are identified by APILABPLUS (Version3.3.3, Bio-Merieux, France) program and standard.
The fermentation process of the Lactobacillus pentosus are as follows: Lactobacillus pentosus is pressed to the inoculation for being 1:6 with the volume ratio of culture medium
Ratio is seeded in MRS fluid nutrient medium, and 18~36h is cultivated in 37 DEG C of incubators, fermentation liquid 5ml is taken to be inoculated with the MRS into 45ml
Expand 18~36h of culture in fluid nutrient medium, under same condition of culture, the 50ml fermentation liquid after taking fermentation is inoculated with into 450ml's
In MRS fluid nutrient medium, continue under same condition of culture expand culture 18~36h, then by culture solution 4000g be centrifuged 5~
8min takes 200ml supernatant to be concentrated 7 times to sticky paste, obtains active-fermented broth used in the present invention.
The composition of the MRS fluid nutrient medium: Ruan peptone 10.0g, beef extract 10.0g, yeast extract 5.0g,
Glucose 20.0g, polysorbate80 1.0g twist lemon acid disodium 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate
0.05g, potassium dihydrogen phosphate 2.0g, distilled water 1L, pH are 6.5 ± 0.2,121 DEG C of sterilizing 20min.
The cultural method of phytophthora blight of pepper: the phytophthora blight of pepper test tube slant that will be activated is cleaned twice, most with physiological saline
Bacterium solution is diluted to 2 × 10 afterwards8A/mL or so, or with spectrophotometric determination, the light transmittance under 650nm is 20%.It saves backup.
Carry out inhibition test to phytophthora blight of pepper using Odontothrips loti: (1) prepare lower layer's culture medium: 30g rye adds distillation
Water 250ml, then 20-30min is boiled in heating, then adds sucrose 10g, agar powder 7.5g is added water to after it is completely dissolved
500ml, 121 DEG C of sterilizing 15min dispense lower layer's culture medium under aseptic condition in sterilized culture dish；(2) preparation upper layer training
It supports base: taking 50 DEG C of lower layer rye culture medium 200ml, Phytophthora capsici bacteria suspension 10ml is added, is uniformly mixed spare.50 DEG C of training
Feeding base can by by the culture medium of high-temperature sterilization it is slightly cooling after be placed in 50 DEG C of thermostat water bath and keep the temperature 30min and prepare.
(3) cultivate: the upper layer culture medium that 5ml is mixed with phytophthora blight of pepper is added in lower layer's culture dish after solidification, back and forth by culture dish
Tilt, is uniformly distributed bacteria-containing upper layer, 4 Oxford cups is spaced apart in each culture dish after the solidification of upper layer culture medium, so
Afterwards with sterile dropper or liquid relief rob by the Lactobacillus pentosus of preparation (Lactobacillus Pentosus) test article solution is added to
In Oxford cup, liquid level is flushed with cup face.Then carefully culture dish is moved in 37 DEG C of insulating boxs and is cultivated 5--7 days.Observe and record training
Antibacterial circle diameter in ware is supported, each test sample sets 3 repetitions, and not connect the MRS fluid nutrient medium of bacterial strain as control, obtains
Test sample evaluates sample to the inhibiting effect of phytophthora blight of pepper with inhibition zone size to the inhibiting rate of phytophthora blight of pepper.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) fermentation liquid preparation inhibit capsicum epidemic disease
Application in mould biological agent.
The beneficial effects of the present invention are: avoiding crop due to using bio-fermented liquid, vegetables use chemical pesticide
Caused chemical pesticide residual, ensure that the edible safety of crop, vegetables.Also mitigate the accumulation of chemical pesticide in the soil
Amount, it is therefore prevented that pollution of the chemical pesticide to soil.Microbes material will not generate pollution in the soil.Make the tellurian mankind
And biology being capable of normal growth and procreation from generation to generation.[plant not fallen ill × (1- disease incidence %) ÷ investigates plant × morbidity to inhibiting rate
Rate % × 100%] it can reach 87%, Lactobacillus pentosus (Lactobacillus Pentosus) fermented supernatant fluid is to Phytophthora capsici
Bacterium significantly inhibits, and can be used in the biological agent of preparation prevention and treatment phytophthora blight of pepper.
Detailed description of the invention
Fig. 1 is the morphosis figure of Lactobacillus pentosus, and A is that Lactobacillus pentosus cultivates 5-7d's on MRS culture medium in figure
Bacterium colony figure, B are × 40 times of micrographs, and C is × 100 times of micrographs；Fig. 2 is Lactobacillus pentosus to phytophthora blight of pepper bacteriostatic experiment
Result figure, in figure: A is the supernatant being concentrated after 7 times, and B is unconcentrated supernatant, and C is the MRS fluid nutrient medium for not connecing bacterial strain
Control；Fig. 3 is Hot Pepper Seedling incidence, and a is Hot Pepper Seedling morbidity stem in figure, and b is Hot Pepper Seedling morbidity leaf.
Specific embodiment
Embodiment 1: Lactobacillus pentosus isolating and purifying and identifying
(the Sichuan cuisine wineshop at China Agricultural University east school district east gate is taken from from Pickles, Sichuan Style long-used soup；NaCl concentration is 4.75%, pH
Be 4.19) in isolated, code name sp, specific separation method be use specific separation method for using dilution 108 apply
Cloth plate method (it is separated using MRS fluid nutrient medium, is cultivated in 37 DEG C of incubators, the following institute of culture medium specific formula
Show), by the flat band method purifying that continuously draws lines, using API50CH reagent strip and API50CHL culture medium (Bio-Merieux,
50410) sugared fermentation reaction is carried out to isolated bacterial strain according to the instruction explanation of manufacturer, test result is by APILABPLUS
(Version3.3.3, Bio-Merieux, France) program and standard identification (API50CH qualification result is as shown in Table 1).
The bacterium colony isolated and purified is smaller, is milky, edge rounding, in spherical, and combine by APILABPLUS program and
The API50CH reagent analysis result that standard is identified is it is found that the strain isolated and purified is Lactobacillus pentosus.
1 API50CH reagent analysis result of table
Table 1 Reagent from AP150CH reagents
+: Positive reaction; -: Negative reaction.
Embodiment 2: Lactobacillus pentosus (Lactobacillus Pentosus) to the Inhibition test of phytophthora blight of pepper
1. Lactobacillus pentosusLactobacillus PentosusThe preparation of fermented supernatant fluid
The Lactobacillus pentosus (CGMCC No.1919) of preservation is seeded in MRS fluid nutrient medium (volume ratio 1 of the two:
6) it when, cultivating 18~36 in 37 DEG C of incubators, takes in 5 milliliters of fermentation liquid accesses, 45 milliliters of MRS fluid nutrient mediums, is equally training
Expand culture under the conditions of supporting, 50 milliliters of fermentation liquids after fermentation are accessed in 450 milliliters of MRS fluid nutrient mediums, then is equally being trained
Expand culture under the conditions of supporting.Culture solution is centrifuged 5~8 minutes in centrifugal force for 4000g, supernatant collection is concentrated 7 times and is extremely glued
Paste.
The composition of the MRS fluid nutrient medium: Ruan peptone 10.0g, beef extract 10.0g, yeast extract 5.0g,
Glucose 20.0g, polysorbate80 1.0g twist lemon acid disodium 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate
0.05g, potassium dihydrogen phosphate 2.0g, distilled water 1L, pH are 6.5 ± 0.2,121 DEG C of sterilizing 20min.
2. the preparation of Phytophthora capsici bacteria suspension
The phytophthora blight of pepper test tube slant that will be activated, is washed down, centrifugation with physiology salt, and thallus is again with physiology salt washing one
It is secondary, bacterium solution is finally diluted to 2 × 108A/milliliter or so, or with spectrophotometric determination, the light transmittance under 650nm is 20%.
It saves backup.
3. Lactobacillus pentosus microbial material is to phytophthora blight of pepper bacteriostatic experiment
Inhibition test is carried out to phytophthora blight of pepper using Odontothrips loti: (1) preparing lower layer's culture medium: weighing rye 30g and add distillation
Water 250mL, then heating stirring is boiled 20-30min and is made it dissolve, and adds sucrose 10g, and agar powder 7.5g is completely dissolved to it
After add water to 500mL, 121 DEG C of sterilizing 15min dispense lower layer's culture medium under aseptic condition in sterilized culture dish；(2) it makes
Standby upper layer culture medium: taking 50 DEG C of lower layer rye culture medium 200ml, and Phytophthora capsici bacteria suspension 10ml is added, is uniformly mixed spare.
50 DEG C of culture medium can keep the temperature 30min by being placed in 50 DEG C of thermostat water bath after slightly cooling down the culture medium of high-temperature sterilization
To prepare.(3) cultivate: the upper layer culture medium that 5ml is mixed with phytophthora blight of pepper is added in lower layer's culture dish after solidification, will cultivate
Ware tilts back and forth, is uniformly distributed bacteria-containing upper layer, and 4 Oxfords are spaced apart in each culture dish after the solidification of upper layer culture medium
Cup, then with sterile dropper or liquid relief rob by the Lactobacillus pentosus of preparation (Lactobacillus Pentosus) test sample is molten
Liquid is added in Oxford cup, and liquid level is flushed with cup face.Then carefully culture dish is moved in 37 DEG C of insulating boxs and is cultivated 5--7 days.Observation
Antibacterial circle diameter in culture dish is recorded, each test sample sets 3 repetitions, and is pair not connect the MRS fluid nutrient medium of bacterial strain
According to obtaining test sample to the inhibiting rate of phytophthora blight of pepper, made with inhibition zone size evaluation sample to the inhibition of phytophthora blight of pepper
With.
Microbial material Lactobacillus pentosus is shown in that Fig. 2, antibacterial circle diameter are shown in Table 2 to the result of phytophthora blight of pepper bacteriostatic experiment,
The data obtained in statistical analysis table 1 is it can be concluded that Lactobacillus pentosusLactobacillus PentosusFermented supernatant fluid is to capsicum
The inhibitory effect of phytophthora is significantly higher than control experiment, and 7 times of Lactobacillus pentosus Lactobacillus Pentosus hairs are concentrated
Ferment supernatant is significantly higher than, and fermented supernatant fluid is not concentrated, illustrates Lactobacillus pentosusLactobacillus PentosusFermentation supernatant
Liquid has inhibiting effect to phytophthora blight of pepper, can be used as the material of prevention and treatment phytophthora blight of pepper.
The detection of 2 diffusion method of tableLactobacillus PentosusFermented supernatant fluid antibacterial circle diameter
Embodiment 3: control efficiency of the Lactobacillus pentosus to phytophthora blight of pepper
Using field experiment detection Lactobacillus pentosus to the control efficiency of phytoph-thora capsici leonian.Transplant 4 leaf phase stalwartness Hot Pepper Seedlings 3
?.Setting are as follows: 1. compare 1: soil is not inoculated with any microorganism；2. compareing 2: soil inoculation phytophthora blight of pepper；3. processing experiment:
Lactobacillus pentosus fermented supernatant fluid (stoste is not concentrated, also undiluted) and phytophthora blight of pepper is added in soil simultaneously.Phytophthora blight of pepper
Inoculum concentration is 500~800 sporangiums of 1g wind desiceted soil；Lactobacillus pentosus fermented supernatant fluid is when capsicum has been planted, from root
Portion uniformly pours into 10ml.Each 6 plants of processing, test is in triplicate.All controls and processing 7,14d investigation, statistics after transplanting
Incidence, calculates disease incidence, and calculation formula are as follows: disease incidence=morbidity strain tree/investigates total strain number × 100%.It the results are shown in Table 3, it is peppery
Green pepper seedling incidence is shown in Fig. 3.
7d after capsicum transplantation of seedlings does not do none plant of morbidity of Hot Pepper Seedling of any processing control 1；It is inoculated with pathogen spore
The disease incidence of capsule control 2 is up to 92 %；The disease incidence of processing is 14 %.12d after transplanting, all processing plant disease incidence
Start to increase, but handle experiment disease incidence and increase at least, is 15 %, reaches the level of signifiance with other processing differences.It is possible this
When processing in antagonistic substance in culturing filtrate utilized or inactivation by the microbial metabolism in plant or soil.Pentose cream
Bacillus fermentation supernatant can inhibit the intrusion of germ in 5~7d after transplanting, it thus is seen that Lactobacillus pentosus generation
The antagonistic substance for thanking to generation plays an important role at the prevention and treatment initial stage of disease.
Control action (%) of 3 different disposal of table to capsicum epidemic disease
Note: data are to test the average value of disease incidence twice in table；It is aobvious up to 0.05 with a line difference lowercase letter indication difference
It writes horizontal.
Claims (3)
1. one plant of Lactobacillus pentosus fermentation liquid, it is characterised in that: the Lactobacillus pentosus fermentation liquid is prepared by the following method:
(1) Lactobacillus pentosus (Lactobacillus Pentosus) China Microbiological bacterium has been preserved on January 16th, 2007
Kind preservation administration committee common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state, postcode 100101), deposit number is CGMCC No.1919；
(2) Lactobacillus pentosus is seeded in MRS fluid nutrient medium by with the volume ratio of culture medium for the inoculative proportion of 1:6,37
18~36h is cultivated in DEG C incubator, is taken fermentation liquid 5ml to be inoculated in the MRS fluid nutrient medium of 45ml, is expanded under same condition of culture
18~36h of big culture, the 50ml fermentation liquid after taking fermentation are inoculated in the MRS fluid nutrient medium of 450ml, continue same culture item
Expand 18~36h of culture under part, culture solution is then centrifuged 5~8min separation bacterium solution and thallus in 4000g, supernatant is taken to be concentrated
7 times, to sticky paste, obtain active-fermented broth used in the present invention.
2. one plant of Lactobacillus pentosus fermentation liquid according to claim 1, it is characterised in that: the MRS fluid nutrient medium
Composition are as follows: Ruan peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, polysorbate80
1.0g twists lemon acid disodium 2.0g, sodium acetate 5.0g, magnesium sulfate 0.1g, manganese sulfate 0.05g, potassium dihydrogen phosphate 2.0g, distilled water
1L, pH are 6.5 ± 0.2,121 DEG C of sterilizing 20min.
3. one plant of Lactobacillus pentosus fermentation liquid described in claim 1 inhibits answering in phytophthora blight of pepper biological agent in preparation
With.
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| CN112592864A (en) * | 2020-12-30 | 2021-04-02 | 山西农业大学 | Pseudomonas linusii N516 and application thereof |
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| CN101015306A (en) * | 2007-02-09 | 2007-08-15 | 中国农业大学 | Leaven for vegetable artificial inoculation fermentation and preparation of fermented vegetable by using same |
| CN102286392A (en) * | 2011-03-01 | 2011-12-21 | 安徽农业大学 | Lactobacillus pentosus, fermentation product of lactobacillus pentosus and application of fermentation product |
| CN103005241A (en) * | 2012-12-31 | 2013-04-03 | 太原师范学院 | Leavening agent for lowering nitrate content of vegetables and method for preparing leavened vegetables by leavening agent |
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| WO2006025167A1 (en) * | 2004-07-29 | 2006-03-09 | Kyoto Prefecture | Plant disease controlling agent and controlling method |
| CN1993052A (en) * | 2004-07-29 | 2007-07-04 | 京都府 | Plant disease controlling agent and controlling method |
| CN101015306A (en) * | 2007-02-09 | 2007-08-15 | 中国农业大学 | Leaven for vegetable artificial inoculation fermentation and preparation of fermented vegetable by using same |
| CN102286392A (en) * | 2011-03-01 | 2011-12-21 | 安徽农业大学 | Lactobacillus pentosus, fermentation product of lactobacillus pentosus and application of fermentation product |
| CN103005241A (en) * | 2012-12-31 | 2013-04-03 | 太原师范学院 | Leavening agent for lowering nitrate content of vegetables and method for preparing leavened vegetables by leavening agent |
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| CN112592864A (en) * | 2020-12-30 | 2021-04-02 | 山西农业大学 | Pseudomonas linusii N516 and application thereof |
| CN112592864B (en) * | 2020-12-30 | 2022-09-27 | 山西农业大学 | Pseudomonas flax N516 and application thereof |
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