CN109112163B - Lactobacillus pentosus fermentation liquor and application thereof in inhibiting phytophthora capsici - Google Patents
Lactobacillus pentosus fermentation liquor and application thereof in inhibiting phytophthora capsici Download PDFInfo
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- CN109112163B CN109112163B CN201810980816.3A CN201810980816A CN109112163B CN 109112163 B CN109112163 B CN 109112163B CN 201810980816 A CN201810980816 A CN 201810980816A CN 109112163 B CN109112163 B CN 109112163B
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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
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
The invention belongs to the technical field of biological control of soil-borne pathogens, and provides lactobacillus pentosus fermentation liquor and application thereof in inhibiting phytophthora capsici. Lactobacillus pentosus: (Lactobacillus Pentosus) Has been preserved in China general microbiological culture Collection center (CGMCC) at 16.1.2007 with the preservation number of CGMCC No. 1919. Avoids chemical pesticide residue caused by using chemical pesticide on crops and vegetables, and ensures the edible safety of the crops and the vegetables. The accumulation of chemical pesticide in the soil is reduced, and the pollution of the chemical pesticide to the soil is prevented. No pollution is generated in the soil. So that the human beings and the organisms on the earth can grow normally and propagate in generations. The inhibition rate can reach 87%, the lactobacillus pentosus fermentation supernatant has an obvious inhibition effect on phytophthora capsici, and can be used for preparing a biological preparation for preventing and treating phytophthora capsici.
Description
Technical Field
The invention belongs to the technical field of biological control of soil-borne pathogens, and particularly relates to lactobacillus pentosus fermentation liquor and application thereof in inhibiting phytophthora capsici.
Background
Soil-borne diseases are diseases and insect pests which propagate, live or overwinter in soil, damage plants on the ground by taking the soil as a transmission medium, and are wide in damage range, high in transmission speed and strong in concealment, so that the loss is huge and difficult to treat. Crop yield losses due to the damage of soil-borne pests can typically reach 20% to 40%, and severe plots can be as high as 60%, or even outcrop. Soil-borne diseases have severely restricted the development of protected areas.
With the adjustment of agricultural structure, the vegetable planting area is enlarged year by year around the country, and the occurrence of various soil-borne diseases of vegetables becomes increasingly serious. Phytophthora capsici is a soil-borne pathogenic bacterium which is widely harmful, has been found in all continents of the world at present, has an ever-expanding host range, can harm 78 plants of 18 families at present, and threatens the mass production of various plants. Phytophthora capsici can produce oospores, and because the oospores have stress resistance and can endure extreme environmental conditions, Phytophthora capsici becomes one of the most difficult diseases to control. Has important practical significance for the research of the prevention and the treatment of the phytophthora capsici.
The application of the soil disinfectant has good effect on soil-borne pathogenic bacteria, namely phytophthora capsici. Especially the application of the high-strength chemical agent soil disinfection technology, greatly reduces the crop yield loss caused by the harm of phytophthora capsici. However, the use of a large amount of chemical pesticide causes excessive pesticide residue on edible parts of crops, and easily causes the problems of soil hardening, soil fertility reduction, environmental pollution and the like. At present, the pesticide effect duration is short when the chemical bactericide is used for prevention and treatment, so that the frequency and the dosage of the pesticide used by farmers are gradually increased, and the accumulation of harmful ingredients in crops, vegetables and soil is also gradually increased. The situation is particularly prominent in the cultivation of greenhouse vegetables, and the damage to the health of human bodies and the ecological environment is great. Therefore, the development of a biological bactericide which has lasting drug effect and is harmless to human bodies and ecological environments to replace chemical pesticides becomes a development direction in China and even world. Currently, a large number of microorganisms with biocontrol potential are being investigated. By screening antagonistic microorganisms, the growth and reproduction of phytophthora capsici can be fundamentally inhibited, so that phytophthora capsici can be inhibited. More importantly, many biocontrol bacteria not only exhibit antagonism against pathogenic bacteria, but also can create a characteristic beneficial micro-ecological environment at the plant rhizosphere [ inula et al, northwest university of agriculture and forestry, 2004, 35 (3): 236-245 ] to promote the absorption and utilization of external nutrients by plants and even to generate metabolites which can promote the growth and the propagation of plants, which is not only a safe and efficient control method, but also required for the sustainable development of green agriculture ecology.
Disclosure of Invention
The invention aims to provide lactobacillus pentosus fermentation liquor and application thereof in inhibiting phytophthora capsici.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) Has been deposited in China general microbiological culture Collection center (CGMCC, Address: no. 3 of Xilu No.1 of Beijing, Chaoyang, Chao code 100101, institute of microbiology, national academy of sciences), the preservation number is CGMCC No. 1919.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) Is separated from Sichuan pickle soup (a certain Sichuan pickle wine house in east of China school of China university, wherein the concentration of NaCl is 4.75%, and the pH is 4.19) with sp as code number, and the specific separation method comprises the following steps: using dilution 108The spread plate method (isolated using MRS liquid medium, cultured in 37 ℃ incubator, the specific formulation of medium MRS is shown below) was purified by the continuous line-drawing plate method, sugar fermentation reactions were performed on the isolated strains using API50CH reagent strips and API50CHL medium (Bio-Merieux, 50410) according to the manufacturer's instructions, and the results were identified by the APILABPLUS (version3.3.3, Bio-Merieux, France) program and standard.
The fermentation method of the lactobacillus pentosus comprises the following steps: inoculating lactobacillus pentosus into an MRS liquid culture medium according to an inoculation ratio of 1:6 in volume ratio to the culture medium, culturing for 18-36 h in an incubator at 37 ℃, inoculating 5ml of fermentation liquid into 45ml of MRS liquid culture medium, performing amplification culture for 18-36 h under the same culture condition, inoculating 50ml of fermentation liquid after fermentation into 450ml of MRS liquid culture medium, continuing amplification culture for 18-36 h under the same culture condition, centrifuging the culture liquid for 5-8 min at 4000g, and concentrating 200ml of supernatant to 7 times to paste to obtain the viscous active fermentation liquid used by the invention.
The MRS liquid medium comprises the following components: 10.0g of raney peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 801.0 g of polysorbate, 2.0g of disodium citrate, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of monopotassium phosphate, 1L of distilled water, pH 6.5 +/-0.2, and sterilization at 121 ℃ for 20 min.
The method for culturing the phytophthora capsici comprises the following steps: washing the test tube inclined plane of activated phytophthora capsici twice with normal saline, and finally diluting the bacterial liquid to 2 x 108About one/mL, or a light transmittance at 650nm of 20% as measured with a spectrophotometer. And (5) storing for later use.
An inhibition test is carried out on phytophthora capsici by adopting an oxford cup method: (1) preparing a lower layer culture medium: adding 30g rye into 250ml distilled water, heating and decocting for 20-30min, adding 10g sucrose and 7.5g agar powder, dissolving completely, adding water to 500ml, sterilizing at 121 deg.C for 15min, and packaging the lower layer culture medium in sterilized culture dish under aseptic condition; (2) preparing an upper culture medium: taking 200ml of the lower layer rye culture medium at 50 ℃, adding 10ml of phytophthora capsici suspension, and uniformly mixing for later use. The medium at 50 ℃ can be prepared by slightly cooling the sterilized medium at high temperature and then incubating in a constant temperature water bath at 50 ℃ for 30 min. (3) Culturing: adding 5ml of upper culture medium mixed with phytophthora capsici into the solidified lower culture dish, tilting the culture dish back and forth to uniformly distribute the upper layer containing bacteria, placing 4 oxford cups in each culture dish at intervals after the upper culture medium is solidified, and then using a sterile dropper or a pipette to prepare the lactobacillus pentosusLactobacillus Pentosus) The test article solution was added to the oxford cup at a level flush with the surface of the cup. The plates were then carefully transferred to a 37 ℃ incubator for 5-7 days. Observing and recording the diameter of the inhibition zone in the culture dish, setting 3 times of the observation zone for each test sample, and using MRS liquid of the unanswered strainThe culture medium is used as a control, the inhibition rate of the test sample on the phytophthora capsici is obtained, and the inhibition effect of the sample on the phytophthora capsici is evaluated according to the size of the inhibition zone.
Lactobacillus pentosus of the present invention (Lactobacillus Pentosus) Application of fermentation liquor in preparation of a biological agent for inhibiting phytophthora capsici.
The invention has the beneficial effects that: because the biological fermentation liquor is used, chemical pesticide residues caused by using chemical pesticides for crops and vegetables are avoided, and the edible safety of the crops and the vegetables is ensured. The accumulation of chemical pesticide in the soil is also reduced, and the pollution of the chemical pesticide to the soil is prevented. The microbial material does not produce pollution in soil. So that the human beings and the organisms on the earth can grow normally and propagate in generations. Inhibition [ non-diseased plant X (1-morbidity%) + ] survey plant X morbidity% × 100%]Up to 87%, Lactobacillus pentosus: (Lactobacillus Pentosus) The fermented supernatant has obvious inhibition effect on phytophthora capsici and can be used for preparing a biological agent for preventing and treating phytophthora capsici.
Drawings
FIG. 1 is a structural diagram of the morphology of Lactobacillus pentosus, wherein A is a colony diagram of Lactobacillus pentosus cultured on MRS medium for 5-7d, B is a micrograph multiplied by 40, and C is a micrograph multiplied by 100; FIG. 2 is a graph showing the results of an experiment for inhibiting Phytophthora capsici by Lactobacillus pentosus, wherein: a is supernatant after 7 times of concentration, B is not concentrated supernatant, C is MRS liquid medium control of the unanswered strain; fig. 3 shows the disease condition of pepper seedlings, wherein a is the diseased stems of the pepper seedlings and b is the diseased leaves of the pepper seedlings.
Detailed Description
Example 1: separation, purification and identification of lactobacillus pentosus
The isolated Sichuan pickle stock (obtained from Sichuan pickle vintage in east China of eastern school of China agricultural university; NaCl concentration: 4.75%, pH: 4.19) is separated, and the code is sp, and the specific separation method is a dilution 108 coating plate method (separation is performed by adopting MRS liquid culture medium, culture is performed in an incubator at 37 ℃, and the specific formula of the culture medium is shown in the following table), the separated strain is purified by a continuous line drawing plate method, an API50CH reagent strip and an API50CHL culture medium (Bio-Merieux, 50410) are used for performing sugar fermentation reaction according to the instructions of manufacturers, and the test result is obtained by the program of API ABPLUS (version3.3.3, Bio-Merieux, France) and the standard identification (API50CH identification result is shown in the table I).
The separated and purified bacterial colony is milky white, round in edge and spherical, and the separated and purified bacterial strain is lactobacillus pentosus according to the analysis result of the API50CH reagent identified by the APILABPLUS program and the standard.
TABLE 1 API50CH reagent analysis results
Table 1 Reagent from AP150CH reagents
+: Positive reaction; -: Negative reaction.
Example 2: lactobacillus pentosus: (Lactobacillus Pentosus) Inhibition experiment for Phytophthora capsici
1. Lactobacillus pentosusLactobacillus PentosusPreparation of fermentation supernatant
Inoculating the preserved lactobacillus pentosus (CGMCC No. 1919) into an MRS liquid culture medium (the volume ratio of the two is 1: 6), inoculating 5ml of fermentation liquor into 45ml of MRS liquid culture medium when culturing in an incubator at 37 ℃ for 18-36 ℃, carrying out amplification culture under the same culture condition, inoculating 50ml of fermentation liquor after fermentation into 450ml of MRS liquid culture medium, and carrying out amplification culture under the same culture condition. And (3) centrifuging the culture solution for 5-8 minutes at the centrifugal force of 4000g, and collecting and concentrating the supernatant fluid by 7 times to form a viscous paste.
The MRS liquid medium comprises the following components: 10.0g of raney peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 801.0 g of polysorbate, 2.0g of disodium citrate, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of monopotassium phosphate, 1L of distilled water, pH 6.5 +/-0.2, and sterilization at 121 ℃ for 20 min.
2. Preparation of phytophthora capsici suspension
Washing the test tube slant of activated phytophthora capsici with normal saline, centrifuging and precipitating, washing the thallus with normal saline once, and finally diluting the bacterial liquid to 2 × 108Around one/ml, or 20% at 650nm, as determined by spectrophotometer. And (5) storing for later use.
3. Bacteriostatic experiment of lactobacillus pentosus microbial material on phytophthora capsici
An inhibition test is carried out on phytophthora capsici by adopting an oxford cup method: (1) preparing a lower layer culture medium: weighing 30g of rye, adding 250mL of distilled water, heating, stirring, boiling for 20-30min to dissolve, adding 10g of sucrose and 7.5g of agar powder, adding water to 500mL after the rye is completely dissolved, sterilizing at 121 ℃ for 15min, and packaging a lower layer culture medium in a sterilized culture dish under aseptic condition; (2) preparing an upper culture medium: taking 200ml of the lower layer rye culture medium at 50 ℃, adding 10ml of phytophthora capsici suspension, and uniformly mixing for later use. The medium at 50 ℃ can be prepared by slightly cooling the sterilized medium at high temperature and then incubating in a constant temperature water bath at 50 ℃ for 30 min. (3) Culturing: adding 5ml of upper culture medium mixed with phytophthora capsici into the solidified lower culture dish, tilting the culture dish back and forth to uniformly distribute the upper layer containing bacteria, placing 4 oxford cups in each culture dish at intervals after the upper culture medium is solidified, and then using a sterile dropper or a pipette to prepare the lactobacillus pentosusLactobacillus Pentosus) The test sample solution was added to the oxford cup at a level flush with the cup surface. The plates were then carefully transferred to a 37 ℃ incubator for 5-7 days. Observing and recording the diameter of the inhibition zone in the culture dish, setting 3 repeats for each test sample, taking MRS liquid culture medium of the unanswered strain as a control to obtain the inhibition rate of the test sample on the phytophthora capsici, and evaluating the inhibition effect of the test sample on the phytophthora capsici according to the size of the inhibition zone.
The results of the bacteriostatic experiment of the microbial material lactobacillus pentosus on phytophthora capsici are shown in figure 2, the diameter of the bacteriostatic circle is shown in table 2, and the data obtained in the statistical analysis table 1 can be used for obtaining the lactobacillus pentosusLactobacillus PentosusThe inhibition effect of the fermentation supernatant on phytophthora capsici is obviously higher than that of a control experiment, and 7 times of Lactobacillus Pentosus fermentation is concentratedThe supernatant is significantly higher than the unconcentrated fermentation supernatant, indicating Lactobacillus pentosusLactobacillus PentosusThe fermented supernatant has an inhibiting effect on phytophthora capsici and can be used as a material for preventing and treating phytophthora capsici.
TABLE 2 diffusion assayLactobacillus PentosusDiameter of bacteriostatic zone of fermented supernatant
Example 3: prevention and treatment effect of lactobacillus pentosus on phytophthora capsici
And detecting the control effect of the lactobacillus pentosus on the phytophthora capsici by adopting a field test. 3 robust pepper seedlings in the 4-leaf stage are transplanted. The method comprises the following steps: comparison 1: the soil is not inoculated with any microorganism; ② comparison 2: inoculating phytophthora capsici to soil; treatment experiment: the soil is simultaneously added with lactobacillus pentosus fermentation supernatant (stock solution, not concentrated and not diluted) and phytophthora capsici. The inoculation amount of the phytophthora capsici is 500-800 sporangia in 1g of air-dried soil; when pepper is planted, 10ml of lactobacillus pentosus fermentation supernatant is poured from the root. Each treatment had 6 strains and the experiment was repeated three times. All controls and treatments were investigated and counted for incidence at 7 and 14d after transplantation, and the incidence was calculated as: incidence = diseased trees/total investigated plants × 100%. The results are shown in Table 3, and the disease condition of the pepper seedlings is shown in FIG. 3.
7d after the pepper seedlings are transplanted, none of the pepper seedlings which are not treated and are compared with the pepper seedlings 1 are attacked; the morbidity of the inoculated pathogenic bacteria sporangium control 2 reaches 92 percent; the incidence of treatment was 14%. At 12d after transplantation, the incidence of all treated plants began to increase, but the incidence of treatment experiments increased minimally, 15%, and reached a significant level compared to other treatments. It is possible that at this point the antagonistic substances in the culture filtrate of the treatment have been metabolically utilized or inactivated by the microorganisms in the plant or soil. The lactobacillus pentosus fermentation supernatant can inhibit the invasion of pathogenic bacteria within 5-7 days after transplantation, and therefore, antagonistic substances generated by the metabolism of lactobacillus pentosus play an important role in the early stage of disease control.
TABLE 3 control of Phytophthora capsici (%). by different treatments
Note: the data in the table are the average of the incidence of the two trials; different lower case letters in the same row indicate differences of up to a 0.05 significance level.
Claims (2)
1. The application of lactobacillus pentosus fermentation liquor in preparing a biological preparation for inhibiting phytophthora capsici is characterized in that: the lactobacillus pentosus fermentation liquor is prepared by the following method:
(1) lactobacillus pentosus: (Lactobacillus Pentosus) Has been preserved in China general microbiological culture Collection center (CGMCC) at 16.1.2007 with the preservation number of CGMCC No. 1919;
(2) inoculating lactobacillus pentosus into an MRS liquid culture medium according to an inoculation ratio of 1:6 in volume ratio to the culture medium, culturing for 18-36 h in an incubator at 37 ℃, inoculating 5ml of fermentation liquid into 45ml of MRS liquid culture medium, performing amplification culture for 18-36 h under the same culture condition, inoculating 50ml of fermentation liquid after fermentation into 450ml of MRS liquid culture medium, continuing amplification culture for 18-36 h under the same culture condition, centrifuging the culture liquid for 5-8 min at 4000g, separating bacterial liquid and thallus, and concentrating the supernatant by 7 times to obtain the used active fermentation liquid.
2. The application of the lactobacillus pentosus fermentation liquid in preparing the biological agent for inhibiting phytophthora capsici according to claim 1, wherein the lactobacillus pentosus fermentation liquid comprises the following components in percentage by weight: the MRS liquid culture medium comprises the following components: 10.0g of raney peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 801.0 g of polysorbate, 2.0g of disodium citrate, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of potassium dihydrogen phosphate and 1L of distilled water, wherein the pH value is 6.5 +/-0.2, and the beef is sterilized at 121 ℃ for 20 min.
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| WO2006025167A1 (en) * | 2004-07-29 | 2006-03-09 | Kyoto Prefecture | 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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