CN110129235A - A kind of heat-resisting plant lactobacillus and its application in agricultural production - Google Patents
A kind of heat-resisting plant lactobacillus and its application in agricultural production Download PDFInfo
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- CN110129235A CN110129235A CN201910451266.0A CN201910451266A CN110129235A CN 110129235 A CN110129235 A CN 110129235A CN 201910451266 A CN201910451266 A CN 201910451266A CN 110129235 A CN110129235 A CN 110129235A
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Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- 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
-
- 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
- C12R2001/25—Lactobacillus plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pest Control & Pesticides (AREA)
- General Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dentistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agronomy & Crop Science (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to functional microorganism screening and applied technical fields, specifically provide one plant of heat-resisting plant lactobacillus, and provide its application in agricultural production.For the lactobacillus plantarum screened from methane-generating pit base sludge, deposit number is CCTCC NO:M2019401.The bacterial strain heat resistance is strong, and has stronger production acid and produce protease ability, and fungistatic effect is it is also obvious that can be widely applied to agricultural production.
Description
Technical field
The present invention relates to the screening of functional microorganism and applied technical fields, and in particular to one plant of novel heat-resisting plant cream
Bacillus and its application in agricultural production.
Background technique
Modern composting technology is that developed country is successful in developmental research early 20th century, at present Britain, the U.S., France,
Germany, Japan and other countries have been widely used.The U.S. is made fertilizer there are about 49% sludge every year and imposes on farmland or forest land.France
Also BIOTERRA mechanically composting technique is applied, good result is obtained.China's Traditional compost method be typically all using having additional nutrients and
The method for improving environmental condition, using the indigenous microorganism in composting raw material come degradable organic pollutant, but due to compost initial stage
Indigenous microorganism amount is few, needs certain time that could breed, and various microorganism decomposition velocity contrasts are very big, therefore passes
Often there is the problems such as fermentation time is long, generation stink and fertilizer efficiency is low in system compost.Modern times composting technology generallys use manually at present
The method for being inoculated with the strong microorganism of decomposing organic matter ability.Since the surface area ratio of microorganism is larger, metabolism is vigorous, and number is huge
Greatly, breeding rapidly, plays a leading role to organic substance degradation in Composting Process.
The fermentation process of organic compost can simply be divided into following 4 stages:
1, glow stage
Compost makes initial stage, and the microorganism in compost is based on medium temperature, aerobic type.They start the fermentation of compost
Process, it is vigorous under the conditions of aerobic to decompose easy decomposition of organic matter (such as simple carbohydrate, starch, protein), it generates a large amount of
Heat, be continuously improved compost temperature, 40 DEG C, referred to as glow stage or middle thermophase are risen to from 20 DEG C or so.
2, hot stage
With the raising of temperature, the microorganism of thermophilic gradually replaces mesothermal type and plays a leading role, and temperature is held
It is continuous to rise, it is within these few days being generally up to 50 DEG C or more, into hot stage.
In hot stage, the microorganism of thermophilic is to organic substance (such as cellulose, hemicellulose, fruit complicated in compost
Glue substance etc.) intensive decomposition, thermal accumlation are carried out, compost temperature rises to 60-70 DEG C, or even up to 80 DEG C.Immediately mostly
Number thermophilic microorganisms also mortality or enter dormant state (20 days or more), this to accelerate compost it is decomposed have it is critically important
Effect.Improperly compost, only very short megathermal period, or high temperature is not achieved at all, thus it is decomposed very slow, in half a year or more
State semi-decomposed is also not achieved in long-term.
The Main Stage that the compost megathermal period decomposes as organic macromolecule, and ensure the important stage of compost hazard-free.
Because the matrix of many compost carries the pathogen and weed seed of the mankind, animals and plants.In composting process, by short
The persistently overheating of time can be such that animals and plants pathogen and weed seed inactivates
3, temperature-fall period
After hot stage certain time, cellulose, hemicellulose, pectin substance have largely been decomposed, and are left
Be difficult to decompose complicated ingredient (such as lignin) and the humus that is newly formed, and the activity decrease of microorganism, temperature is gradually reduced.When
When temperature drops to 40 DEG C or less, middle warm nature microorganism becomes dominant species again.
If the morning that temperature-fall period comes, showing that composting condition is not ideal enough, vegetable matter decomposes insufficient.It at this moment can be with
Accumulation material is mixed in turning thoroughly, is allowed to generate second of fever, heating, to promote the decomposed of compost.
4, the decomposed fertilizer conservation stage
After compost maturity, volume-diminished, heap temperature drops to a little higher than temperature, at this moment should compress compost, causes shape of being sick of
State makes organic matter mineralization declines, in favor of fertilizer conservation.
In brief, the fermentation process of organic compost is actually the process of various bacteria metabolisms, breeding.It is micro-
The process that the metabolic processes of biology, that is, organic matter decomposes.Organic matter decomposition will necessarily generate energy, these energy push
Composting process, elevates the temperature, while can also dry wet basis matter.
The major microorganisms type for participating in composting process is bacterium, fungi and actinomyces etc..During these microorganisms have
Warm bacterium and high temperature bacterium.Wherein, lactic acid bacteria (anaerobic type) is a kind of compost commonly bacterium, it is to absorb photosynthetic bacteria saccharomycete
Based on the substances such as the carbohydrate of generation, lactic acid is generated.Lactic acid has very strong sterilizing ability, can effectively inhibit harmful microbe
The sharply corruption of activity and organic matter is decomposed.Lactic acid bacteria can be such that the lignin being not easily decomposed under normality and cellulose etc. becomes
It must be easy to decompose, and eliminate various drawbacks that undecomposed organic matter generates, play shock brigade in organic matter fermentation decomposition
Important function.But the heat resistance of lactic acid bacteria is poor, and different Pseudomonas optimum growth temperatures are substantially at 35~45 DEG C.Therefore, by high temperature
The influence of factor, lactic acid bacteria can generally reduce in compost megathermal period population quantity and activity, thus limit the quick of organic matter
It decomposes.Thus breeding lactic acid bacteria strains resistant to high temperature have particularly important theory significance and wide application prospect.
Summary of the invention
The present invention is to solve prior art problem, provides one plant of heat-resisting plant lactobacillus, and it is raw in agricultural to provide it
Application in production.For the lactobacillus plantarum screened from methane-generating pit base sludge, heat resistance is strong, and has stronger production acid and lay eggs
White enzyme ability, fungistatic effect is it is also obvious that can be widely applied to agricultural production.
One aspect of the present invention provides a kind of lactobacillus plantarum LLH-2 (Lactobacillus plantarum LLH-2),
It is preserved in the China typical culture collection center of Wuhan, China Wuhan University on May 27th, 2019, deposit number is
CCTCC NO:M2019401.
The present invention also provides application of the lactobacillus plantarum LLH-2 in compost.
The present invention also provides application of the lactobacillus plantarum LLH-2 in control of plant disease.
The plant disease includes root rot, southern blight, powdery mildew, anthracnose, shot hole, gray mold, wilt disease, foxiness
Disease or fruit rot in any one.
Further aspect of the present invention provides a kind of biological decomposing agent, includes above-mentioned lactobacillus plantarum LLH-2.
The decomposed Inoculant also include bacillus, coccus, Aspergillus, yeast, Trichoderma, streptomycete, Micropolyspora,
Actinomyces, pseudomonad, any one or two or more of combinations in lactic acid bacteria.
The viable bacteria amount of lactobacillus plantarum LLH-2 is at least 10 in the decomposed Inoculant9CFU/g。
The present invention also provides application of the above-mentioned biological decomposing agent in organic materials compost.
The organic materials includes mushroom dreg, maize peel, peanut shell, feces of livestock and poultry, crop material, municipal sludge, meal
Any one in kitchen rubbish, food processing or pharmacy waste residue or two or more of combinations.
The additive amount of the biology decomposing agent is 0.5-5kg/ tons of materials.
Beneficial effect
The lactobacillus plantarum LLH-2 that the present invention screens has very strong heat resistance, remains under 50-70 DEG C of high temperature a large amount of
Survival and breeding, and the acids such as efficient secretion lactic acid.Compared with the lactic acid production under the conditions of 37 DEG C, the bacterial strain at 50 DEG C and
Up to 85.1% and 65.2% lactic acid production is remained under 70 DEG C of hot conditions respectively.Lactobacillus plantarum LLH-2 also have compared with
Strong production protease ability, 37 DEG C of culture 48h, proteinase activity is up to 97U/mL in fermented supernatant fluid.Inoculated plant in compost
Lactobacillus LLH-2 can significantly improve the activity of microorganism, increase compost temperature rapidly to 68 DEG C, and greatly prolong the megathermal period
Duration is conducive to kill pathogenic microorganism in compost, shortens the compost time, improves compost quality.
In addition, lactobacillus plantarum LLH-2 provided by the invention to Escherichia coli, salmonella, staphylococcus aureus and
The pathomycetes such as the pathogenic bacterias such as Listeria and pine root fungus, sclerotium rolfsii, Streptomyces scabies and brown patch germ, have compared with
Strong antagonism.In field experiment, which is up to 83.0% He to the prevention and treatment efficiency of root rot and brown spot respectively
80.0%, to the prevention and treatment efficiency of southern blight and shot hole more than 55%, it is significantly higher than the prevention and treatment effect of chemicals treatment group carbendazim
Fruit.Therefore, lactobacillus plantarum LLH-2 provided by the invention can be used as biocontrol agent, for preventing and treating frequently seen plants disease, and to ring
Border is friendly, is conducive to the quality for promoting crops, pushes transformation of the traditional agriculture to the ecological agriculture, green agriculture, realizes agricultural
Health, sustainable development.
Detailed description of the invention
Fig. 1 is the temperature variation of heap body in Composting of Chicken Manure.
Specific embodiment
The present invention is further explained combined with specific embodiments below.For specific method or material used in embodiment
Material, those skilled in the art can carry out conventional replacement according to existing technology and select on the basis of the technology of the present invention thinking
It selects, is not limited solely to the specific record of the embodiment of the present invention.Equipment selected by the present invention and reagent can be selected from commercially available any
It is a kind of.
The screening of 1 high temperature resistant lactic acid bacteria of embodiment and with identification
The screening of 1.1 high temperature resistant lactic acid bacterias
Sample: Qingdao of Shandong province Pingdu Liaolan town farm methane-generating pit base sludge.
By mud sample through 70 DEG C of high temperature enrichment cultures, with the pure bacterial strain of the isolated 55 plants of high temperature resistants of gradient dilution method.It will
Isolated bacterial strain be seeded to respectively plus 0.5% calcium carbonate MRS plate (peptone 10g, yeast extract 5g, beef extract 10g,
Glucose 20g, dipotassium hydrogen phosphate 2g, ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganese sulfate 0.25g, Tween 80 mL,
Agar 15g, distilled water 1.0L composition, pH6.5 ± 0.2) on, 37 DEG C of culture 48h are separately cultured the bacterial strain of transparent circle, obtain 3
Strains of lactic acid bacteria is respectively designated as LLH-1, LLH-2, LLH-3；
Above-mentioned 3 strains of lactic acid bacteria is inoculated in respectively in 50mL MRS broth bouillon, respectively in 37 DEG C, 50 DEG C and 70 DEG C items
Under part, 48h is respectively cultivated, measures the content of lactic acid in culture medium respectively with HPLC, with the pH of pH meter measurement culture medium, concrete outcome
As shown in table 1.
1 high temperature resistant lactic acid bacteria of table acid producing ability under different cultivation temperatures compares
From the results shown in Table 1, in 3 strains of lactic acid bacteria that the present invention screens, LLH-2 bacterial strain is in 37 DEG C, 50 DEG C, 70
The equal highest of lactic acid production under the conditions of DEG C, and after fermenting culture medium pH value it is also minimum, to illustrate that the comprehensive of LLH-2 bacterial strain produces
Sour ability is most strong.
Moreover, LLH-2 bacterial strain is protected respectively under 50 DEG C and 70 DEG C of hot conditions compared with the lactic acid production under the conditions of 37 DEG C
Up to 85.1% and 65.2% lactic acid production is stayed, the pH value of culture medium is respectively 3.55 and 4.07 after fermentation.To illustrate,
The LLH-2 bacterial strain that the present invention screens has very strong heat resistance, and large number of viable and numerous is remained under 50-70 DEG C of hot conditions
It grows, and the acids such as efficient secretion lactic acid, effect highly significant.
The identification of 1.2 bacterial strains
The bacterium colony of LLH-2 bacterial strain is milky, neat in edge；Gram's staining is positive, and cellular morphology is rod-short, nothing
Gemma；Upgrowth situation is good under the conditions of 15 DEG C -70 DEG C.LLH-2 bacterial strain is cultivated 24 hours in shaking flask MRS broth bouillon
Afterwards, number of viable is more than 1,000,000,000/mL.
The LLH-3 bacterial strain that above-mentioned screening obtains is identified using the method for molecular biology, measures its 16s rDNA
Sequence SEQ ID NO:1.SEQ ID NO:1 is subjected to blast comparison in GenBank nucleic acid database, finds itself and plant
The 16s rDNA sequence similarity of lactobacillus is up to 98.56%.
SEQ ID NO:1 is as follows:
aaagatggcttcggctatcacttctggatggtcccgcggcgtattaggtagatggtggggtaacggct
caccatggcaaagatacgtagccgacctgagagggtaatcggccacattgggactgagacacggcccaaactccta
cggggggcagcagtagggaattttccaaaatggacgaaagtctgatggagcaacgccgcgtgagtgaagaagggtt
tcggctcgtaaaactcttttgttaaagaagaacatatctgagagtaaattttcaggtattgacggtatttaaccag
aaagccacggctaactacctgccagcaggcggggtaattcgtaggtggcaagggttgtcaggatttattgggcgta
aagcgagcgcaggcggttttttaagtctgatgtgaaagcctttggctcaaccgaagaagtgcatcggaaactggga
aaattgaatgcagaagaggacagtggaactctatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtg
gcgaaggcggctgtctggtctgtaactgacgctgaggctcgaaagtatgggtagcaaacaggattagataccctgg
tagtccataccgtaaacgatgaatgctaagtgttggagggtttccgcccttcagtgctgcagctaacgcattaagc
attccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatg
tggtttaattcgaagctacgcgaagaaccttaccaggtcttgacatactatgcaaatctaagagattagacgttcc
cttcggggacatggatacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaac
gagcgcaacccttattatcagttgccagcattaagttgggcactctggtgagactgccggtgacaaaccggaggaa
ggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagt
tgcgaactcgcgagagtaagctaatctcttaaagccattctcagttcggattgtaggctgcaactcgcctacatga
agtcggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtca
caccatgagagtttgtaacacccaaagtcggtggggtaaccttta
In conjunction with the colonial morphology and 16srDNA comparison result of LLH-2 bacterial strain, applicant determined that LLH-2 bacterial strain is plant cream
Bacillus (Lactobacillus plantarum), is named as lactobacillus plantarum LLH-2 (Lactobacillus plantarum
LLH-2)。
Applicant is on May 27th, 2019 by above-mentioned lactobacillus plantarum LLH-2 (Lactobacillus plantarum
LLH-2 it) is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:
M2019401。
2 lactobacillus plantarum LLH-2 of embodiment lays eggs white enzyme activity determination
Lactobacillus plantarum LLH-2 is accessed into MRS fluid nutrient medium (peptone 10g, yeast extract 5g, beef extract 10g, grape
Sugared 20g, dipotassium hydrogen phosphate 2g, ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganese sulfate 0.25g, Tween 80 lmL, distillation
Water 1.0L composition, pH6.5 ± 0.2) in, it is cultivated at 37 DEG C for 24 hours, continuous switching two generations of activation, with the access of 1% (/v/v) inoculum concentration
In 200mL MRS fluid nutrient medium, 37 DEG C of culture 48h；4 DEG C, 8000r/min is centrifuged 10min, collects supernatant, and use is following
Method detects proteinase activity in supernatant.
The results show that proteinase activity is up to 97U/ in the lactobacillus plantarum LLH-2 fermented supernatant fluid that the present invention screens
ML, unexpected technical results have been achieved.
(1) enzyme activity defines: 37 DEG C, decomposing enzyme amount needed for bovine serum albumin generates the tryptophan of 1 μm of ol per minute is
One enzyme-activity unit.
(2) enzyme activity determination method: taking 50 μ L concentration is 1% (w/v) bovine serum albumin (BSA), 450 μ L of enzyme solution, with concentration
1.5mL sodium acetate buffer for 0.1mol/L, pH 7.0 mixes, and keeps the temperature 5min at 37 DEG C.It is 10% with 0.5mL concentration
Trichloroacetic acid terminates reaction, and light absorption value is measured at 280nm.
Blank control pipe: enzyme solution similarity condition operation as above is replaced with distilled water.
Enzyme activity formula: enzyme activity (U/mL)=(k × w) (/v × T), wherein K is enzyme solution extension rate；W is the color ammonia generated
Acid amount (μm ol)；V is reaction enzymes liquid product (mL)；T is reaction time (min).
Application of the 3 lactobacillus plantarum LLH-2 of embodiment in compost
1, compost material is chicken manure and sawdust, is taken respectively from chicken farm and the wood-working factory of Qingdao City's Pingdu.
2, experimental group is arranged:
(1) blank control group: inoculation sterile water；
(2) CGMCC1.572 processing group: inoculated plant lactobacillus CGMCC1.572；
(3) LLH-2 processing group: inoculated plant lactobacillus LLH-2.
According to the property of chicken manure and sawdust, moisture regulation to 60% or so, C/N ratio is adjusted between 25~30, sufficiently
After mixing, it is fitted into the foam box that volume is 4L.
Before compost inoculation, first by lactobacillus plantarum fermentation liquid ((108-109Cfu/ml)) with the 5 ‰ of material weight in wet base and corn
Face is mixed well in the ratio that volume mass is 1: 1, is seeded in compost material when heap temperature rises to 40 DEG C or more.It is empty
White control group replaces fermentation liquid to mix addition with maize flour with equivalent sterile water.
By equipped with compost material foam box be placed in 18 DEG C of room temperature or so of laboratory carry out it is decomposed.Respectively in every the sky
For noon 10:30 and lower 16:30 to temperature measuring is carried out inside heap body, each experimental group respectively takes 3 point measurements, and records ring at that time
Border temperature takes the average value of measured value in 1d to be used as same day temperature value.The result is shown in Figure 1.
It will be seen from figure 1 that heap temperature experienced rapid increase, maintain, the process then declined.Heap temperature is in heap
Fertilizer rises to 40 DEG C or so on the 2nd day after starting.After inoculated plant lactobacillus, LLH-2 processing group enters the megathermal period on day 3, temperature
Up to 70 DEG C of degree；CGMCC1.572 processing group also enters the megathermal period on day 3, but temperature only has 60 DEG C；And blank control group the 3rd
It when, heap temperature is only 54 DEG C, and the 4th talent enters the megathermal period, and heap temperature is 57 DEG C, and substantially less than lactobacillus plantarum is handled
Group.
Compost the 7th day, LLH-2 processing group heap temperature maintained 53 DEG C, the decline of CGMCC1.572 processing group heap temperature
To 47 DEG C, and blank control group heap temperature drops to 44 DEG C, substantially less than LLH-2 processing group.LLH-2 processing group heap body is most
High-temperature reaches 70 DEG C, 10 DEG C higher than CGMCC1.572 processing group maximum temperature (60 DEG C), than blank control group maximum temperature (57
DEG C) 13 DEG C high；And LLH-2 processing group megathermal period Duration Ratio CGMCC1.572 processing group is 1 day more, and it is more than blank control group
2 days.
The above results illustrate that being inoculated with high-temperature resistant strain lactobacillus plantarum LLH-2 provided by the invention in compost can be significant
The activity for improving microorganism, increases rapidly compost temperature, and duration megathermal period is longer, and it is micro- to be conducive to kill cause of disease in compost
Biology shortens the compost time, improves compost quality.Therefore, it is decomposed to can be used as biology by lactobacillus plantarum LLH-2 provided by the invention
Agent is widely used in maize peel, peanut shell, mushroom dreg, feces of livestock and poultry, crop material, municipal sludge, kitchen garbage, food and adds
The organic materials compost such as work and pharmacy waste residue, significant effect.
The measurement of 4 lactobacillus plantarum LLH-2 bacteriostasis of embodiment
1, the rejection ability of bacterium is measured
1) prepared by lactobacillus suspension: lactobacillus plantarum LLH-2 being inoculated in 100mL MRS fluid nutrient medium, 37 DEG C of standings
Cultivate 48h；
2) prepared by pathogenic bacteria bacterium solution: respectively by Escherichia coli, salmonella, staphylococcus aureus and Listeria strain
It is inoculated in nutrient broth medium, 37 DEG C of shaking table cultures are stayed overnight；
3) bacteriostatic experiment-double-layer plate, Odontothrips loti: (50 DEG C or so) of every 5mL bactericidal nurishing agar medium plus 100 μ
L pathogenic bacteria bacterium solution (bacterium amount 106The order of magnitude), hypsokinesis is mixed down to nutrient agar panel and is made into double-layer plate, is being cultivated after solidification
Oxford cup is placed on base, the cultured lactobacillus plantarum LLH-2 bacterium solution of 200 μ L is added into Oxford cup, is put after bacterium solution diffusion
Enter and cultivate 20h in 37 DEG C of incubators, observes antibacterial circle diameter.It the results are shown in Table 2.
Inhibiting effect of the 2 lactobacillus plantarum LLH-2 of table to pathogenic bacteria
| Pathogenic bacteria | Escherichia coli | Salmonella | Staphylococcus aureus | Listeria |
| Antibacterial circle diameter | 28mm | 25mm | 27mm | 28mm |
As can be seen from the above table, the lactobacillus plantarum LLH-2 that the present invention filters out is to salmonella, staphylococcus aureus
There is very strong inhibiting effect with Listeria, especially most strong to the inhibiting effect of Escherichia coli and Listeria, inhibition zone is straight
Diameter reaches 28mm.
2, the rejection ability of pathomycete is measured
(1) pathomycete is cultivated:
In aseptic operating platform, it is inoculated with peanut root rot bacterium, sclerotium rolfsii, Streptomyces scabies and brown patch germ respectively to PDA
In culture medium, 28 DEG C of inversions are cultivated 3 days.
(2) inoculation face-off bacterium:
It grows when pathomycete to when accounting for about culture dish 1/3, inoculated plant lactobacillus LLH-2 is being distinguished at fungi 2cm,
Each cause is measured respectively after continuation is cultivated 3 days under the conditions of 37 DEG C using the fungi of non-inoculated plant lactobacillus LLH-2 as control
The colony radius of sick fungi calculates bacteriostasis rate.
Bacteriostasis rate=[(control fungi growth radius-processing fungi grows radius)/control fungi grows radius] ×
100%.
The results show that lactobacillus plantarum LLH-2 has apparent antagonism to above-mentioned four pathomycetes of cultivating peanut.Its
In, LLH-2 is most strong to the inhibiting effect of peanut root rot bacterium and brown spot, and inhibiting rate is up to 84.5% and 80.7% respectively；It is right
The inhibiting effect of Sclerotium rolfssi and shot hole is relatively weak, and inhibiting rate is respectively 58.7% and 60.8%.
Application of the 5 lactobacillus plantarum LLH-2 of embodiment in peanut disease prevention and treatment
1, place is tested:
The peanut continuous cropping field in dune ridge village after the Pingdu of Qingdao, peanut root rot, southern blight, shot hole and brown spot occur tight
Weight.
2, experimental design:
It is randomly provided test block, each test block is the rectangular region of 6m × 10m, and keeps 3 between each test block
Meter or more interval.The line-spacing of peanut is 40cm, spacing in the rows 20cm.Three parallel laboratory test areas are arranged in each experimental group.
(1) blank control group: clear water；
(2) fungicide processing group: 50% 800 times of carbendazim liquid；
(3) lactobacillus plantarum CGMCC1.572 processing group: respectively sowing time, after planting 15d, 30d, 45d utilize plant
Lactobacillus CGMCC1.572 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 50mL of dilution every time；
(4) lactobacillus plantarum LLH-2 processing group: respectively sowing time, after planting 15d, 30d, 45d utilize lactobacillus plantarum
LLH-2 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 50mL of dilution every time.
Other field management investigate incidence after 75 days, as a result as shown in Table 3-6 with normal production.
Root rot grade scale:
0 grade: equal disease-free spot on stem foot and main fibrous root；
1 grade: having a small amount of scab on stem foot and main root；
3 grades: scab is more on stem foot and main root, and lesion area accounts for the 1/4~1/2 of stem foot and the root gross area；
5 grades: scab is more and big on stem foot and main root, and lesion area accounts for the 1/2~3/4 of stem foot and the root gross area；
7 grades: scab in flakes, is formed around stem phenomenon, but root system is not dead on stem foot and main root；
9 grades: root system necrosis, plant above ground portion wilts or death.
Southern blight grade scale:
0 grade: plant is asymptomatic；
1 grade: only generating scab in basal part of stem；
2 grades: basal part of stem generates contracting symptom of hanging, and systemic symptom (withered, dead, wilting is showed below the one third of whole strain
Deng)；
3 grades: 2/3rds or less whole strains show systemic symptom；
4 grades: 2/3rds or more representation system symptoms of whole strain.
Shot hole grade scale:
0 grade: healthy plant
1 grade: occurring small scab on top tender leaf and carpopodium
2 grades: occurring small scab on tender leaf, carpopodium, stem
3 grades: tender leaf edge upsweeps, and occurs scab shape on peanut stem and carpopodium
4 grades: carpopodium, stem severe bends, plant show calcination shape
Brown spot grade scale:
0 grade: disease-free symptom；
1 grade: aggrieved blade area accounts for 1/10 or less investigation blade area；
2 grades: aggrieved blade area accounts for 1/4 or less investigation blade area；
3 grades: aggrieved blade area accounts for 1/2 or less investigation blade area；
4 grades: aggrieved blade area accounts for 1/2 or more of investigation blade area, fallen leaves.
Diseased plant rate=morbidity strain number/total strain number × 100%
Disease index=∑ (morbidity grade typical value × diseased plant number at different levels) × 100/ (fall ill generation by investigation total strain number × superlative degree
Tabular value)
Prevent and treat efficiency=[(control disease index-processing disease index)/control disease index] × 100%
3 peanut root rot control efficiency of table compares
4 peanut sclerotium rolfsii control efficiency of table compares
5 Peanut Scab control efficiency of table compares
6 cercospora brown spot of peanut control efficiency of table compares
It can be seen that lactobacillus plantarum LLH-2 provided by the invention to peanut root-rot from the field experiment data of table 3-6
Disease, southern blight, shot hole and brown spot have apparent control efficiency, wherein being up to the prevention and treatment efficiency of root rot and brown spot
With 83.0% and 80.0%, the prevention and treatment efficiency to southern blight and shot hole is more than 55%, is significantly higher than chemicals treatment group carbendazim
Control efficiency.Although and commercially available lactobacillus plantarum CGMCC1.572 is to peanut root rot, southern blight, shot hole and brown spot
Also there is certain control efficiency, but prevent and treat efficiency and be only below 10%, be far below lactobacillus plantarum LLH-2 of the present invention.
The above results show that lactobacillus plantarum LLH-2 provided by the invention will show the control efficiency of peanut Common Diseases
It writes and is better than traditional chemical bactericide, and is environmentally friendly, be conducive to the quality for promoting crops, can be widely applied to green agriculture
In industry production.
Sequence table
<110>Qingdao force favour biotech inc
<120>a kind of heat-resisting plant lactobacillus and its application in agricultural production
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1253
<212> DNA
<213>lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
aaagatggct tcggctatca cttctggatg gtcccgcggc gtattaggta gatggtgggg 60
taacggctca ccatggcaaa gatacgtagc cgacctgaga gggtaatcgg ccacattggg 120
actgagacac ggcccaaact cctacggggg gcagcagtag ggaattttcc aaaatggacg 180
aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta aaactctttt 240
gttaaagaag aacatatctg agagtaaatt ttcaggtatt gacggtattt aaccagaaag 300
ccacggctaa ctacctgcca gcaggcgggg taattcgtag gtggcaaggg ttgtcaggat 360
ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag cctttggctc 420
aaccgaagaa gtgcatcgga aactgggaaa attgaatgca gaagaggaca gtggaactct 480
atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 540
ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta gataccctgg 600
tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct tcagtgctgc 660
agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa 720
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 780
cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct tcggggacat 840
ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 900
caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct ggtgagactg 960
ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1020
ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag agtaagctaa 1080
tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1140
tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1200
gcccgtcaca ccatgagagt ttgtaacacc caaagtcggt ggggtaacct tta 1253
Claims (10)
1. a kind of lactobacillus plantarum, which is characterized in that the deposit number of the lactobacillus plantarum is CCTCC NO:M2019401.
2. application of the lactobacillus plantarum described in claim 1 in organic materials compost.
3. application of the lactobacillus plantarum described in claim 1 in control of plant disease.
4. application as claimed in claim 3, which is characterized in that the plant disease includes root rot, southern blight, white powder
Any one in disease, anthracnose, shot hole, gray mold, wilt disease, brown spot or fruit rot.
5. a kind of decomposed preparation of biology, which is characterized in that the decomposed preparation of biology includes plant cream described in claim 1
Bacillus.
6. the decomposed preparation of biology as claimed in claim 5, which is characterized in that the decomposed preparation of biology also includes gemma bar
Bacterium, coccus, Aspergillus, yeast, Trichoderma, streptomycete, Micropolyspora, actinomyces, pseudomonad, any one in lactic acid bacteria
Or two or more of combinations.
7. such as the decomposed preparation of biology described in claim 5 or 6, which is characterized in that plant cream in the decomposed preparation of biology
Viable bacteria Liang≤10 of bacillus9 CFU/g。
8. application of any decomposed preparation of biology of claim 5-7 in organic materials compost.
9. application as claimed in claim 8, which is characterized in that the organic materials includes mushroom dreg, maize peel, peanut
Any one or two kinds in shell, feces of livestock and poultry, crop material, municipal sludge, kitchen garbage, food processing or pharmacy waste residue or
A variety of combinations.
10. application as claimed in claim 8 or 9, which is characterized in that the additive amount of the biology decomposing agent is 0.5-5kg/ tons
Material.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269072A (en) * | 2020-03-26 | 2020-06-12 | 甘肃葫阳生物科技有限公司 | Organic fertilizer |
| CN112063548A (en) * | 2020-08-19 | 2020-12-11 | 杭州医学院 | Lactobacillus plantarum capable of producing protease and application thereof |
| CN112430162A (en) * | 2020-12-14 | 2021-03-02 | 济南达信生物科技有限公司 | Lactobacillus and application thereof in bio-organic fertilizer |
| CN113564070A (en) * | 2021-07-16 | 2021-10-29 | 江苏稼润农业开发有限公司 | Biological control enzyme fermentation strain system for edible fungi, fermentation method and application of biological control enzyme fermentation strain system in rice seedling stage for disease resistance |
| CN114436698A (en) * | 2022-02-17 | 2022-05-06 | 青岛力力惠生物科技股份有限公司 | Liquid compound microbial fertilizer and application thereof in agricultural production |
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|---|---|---|---|---|
| CN101974467A (en) * | 2010-10-26 | 2011-02-16 | 郑州大学 | Lactobacillus plantarum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111269072A (en) * | 2020-03-26 | 2020-06-12 | 甘肃葫阳生物科技有限公司 | Organic fertilizer |
| CN112063548A (en) * | 2020-08-19 | 2020-12-11 | 杭州医学院 | Lactobacillus plantarum capable of producing protease and application thereof |
| CN112430162A (en) * | 2020-12-14 | 2021-03-02 | 济南达信生物科技有限公司 | Lactobacillus and application thereof in bio-organic fertilizer |
| CN113564070A (en) * | 2021-07-16 | 2021-10-29 | 江苏稼润农业开发有限公司 | Biological control enzyme fermentation strain system for edible fungi, fermentation method and application of biological control enzyme fermentation strain system in rice seedling stage for disease resistance |
| CN113564070B (en) * | 2021-07-16 | 2023-11-28 | 江苏稼润农业开发有限公司 | Edible fungus bio-fermentation element fermentation strain, fermentation method and application of edible fungus bio-fermentation element fermentation strain in rice seedling stage disease resistance |
| CN114436698A (en) * | 2022-02-17 | 2022-05-06 | 青岛力力惠生物科技股份有限公司 | Liquid compound microbial fertilizer and application thereof in agricultural production |
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