CN110117566A - A kind of complex micro organism fungicide increase soil fertility and its application - Google Patents
A kind of complex micro organism fungicide increase soil fertility and its application Download PDFInfo
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- CN110117566A CN110117566A CN201910451257.1A CN201910451257A CN110117566A CN 110117566 A CN110117566 A CN 110117566A CN 201910451257 A CN201910451257 A CN 201910451257A CN 110117566 A CN110117566 A CN 110117566A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- 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
- C12N1/14—Fungi; Culture media therefor
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- 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
Abstract
The present invention relates to functional microorganism screening and applied technical fields, specifically provide a kind of complex micro organism fungicide increase soil fertility, and provide preparation method and application.The complex micro organism fungicide cooperates with facilitation by bacillus subtilis CCTCC NO:M2019402 and multiple-microorganism, the content that can effectively improve available phosphorus in soil significantly improves soil fertility, increases crop yield, it can also inhibit the generation of pest and disease damage simultaneously, application prospect is extensive.
Description
Technical field
The present invention relates to the screening of functional microorganism and applied technical fields, and in particular to it is a kind of increase soil fertility answer
Close microbial bacterial agent and its application.
Background technique
Chemical fertilizer is that agricultural development is made that major contribution, but chemical fertilizer also has two big disadvantages: first is that due to big
The problems such as amount uses chemical fertilizer, causes soil hardening, acidification, organic matter shortage, microorganism reduction, productivity decline.Two
It is that general chemistry utilization rate of fertilizer is low.According to statistics, Chinese this season utilization rate of nitrogen fertilizer is only 20%~35%, and the utilization rate of phosphate fertilizer is
10%~20%, the utilization rate of potash fertilizer is 30%~60%.Utilization rate of fertilizer is low not only to result in waste of resources, and returns environment and brings
Serious pollution.
Microorganism fertilizer has many advantages, such as to improve soil, increases yield, improves quality, protection environment compared with common fertilizer.
Specific functional microorganism promotes conversion, the raising crop nutrition of substance in soil by the vital movement of itself in microorganism fertilizer
Growth that is horizontal, promoting and assist nutrient absorption, stimulation regulation crop, prevention and treatment harmful microorganism etc., Lai Zengjia crop yield and
Improve crop quality.
Microbial manure is by certain beneficial microbes through bio-feritlizer, also known as bacterial manure, bacterium made of a large amount of artificial cultures
Agent, Inoculant.Its principle be increase the content of nitrogen or available phosphorus, potassium in soil using the vital movement of microorganism, or
The substance that crops some in soil cannot directly be utilized is converted into the nutriment that can be absorbed and used, or improves crop
Excitor substance is produced, or the activity of phytopathogen is inhibited to improve the nutritional condition of crop to increase soil fertility, is improved
Crop yield.It is broadly divided into following a few classes according to its fertilizer effect: (1) increases the bacterium of soil nitrogen and crop nitrogen nutrition
Fertilizer, such as rhizobia fertilizer, azotobacteria fertilizer, Azotica；(2) bacterial manure of the soil organism, such as organic phosphorous bacterial fertilizer are decomposed, it is comprehensive
Conjunction property bacterial manure；(3) bacterial manure for decomposing soil slightly solubility minerals, such as inorganic phosphorus bacteria fertilizer, B. mucilaginocus bacterial manure；(4) stimulation is planted
The bacterial manure of object growth, such as antibiotic bacteria fertilizer；(5) increase the bacterial manure that root of the crop absorbs nutritional capacity, such as VA Mycorrhizal Fungi fertilizer.
The research of China's microorganism fertilizer starts from the research to rhizobium.At the beginning of the sixties, the Chen Hua last of the ten Heavenly stems etc. will be from Chinese milk vetch
Microbial inoculum is made in the rhizobium filtered out, and has carried out the demonstration and popularization of large area.Yin Xinyun etc. is separated using from rhizosphere of alfalfa
The actinomyces arrived, manufactured " 5406 bacterial manure " are used widely, and the further development of China's microorganism fertilizer is promoted.With skill
The progress of art, microorganism fertilizer are increasingly becoming the important with one of fertilizer of the ecological demonstration zone, green and organic agriculture production base.Using
Range from leguminous plant to cereal crops again to plants such as vegetables, fruit tree, flowers, in agricultural production in occupation of more and more important
Position.
So far, more than 60 country promotes and applies microbial manure in the world, these countries and regions are mainly divided
Cloth is in Asia, South America, Europe And Africa etc..The microbial manure developed to comply with agricultural sustainable development has extremely light
Bright application prospect, but the performance of its function is perfect not yet, and presently commercially available product category is relatively fewer, and uses effect
Fruit is unstable, and it is larger to promote difficulty.Therefore, the excellent microorganism fungus kind with functions such as phosphorus decomposing, potassium decomposing, fixed nitrogen of breeding, still
It is so the top priority of current microorganism fertilizer product development.
Summary of the invention
The present invention is to solve prior art problem, provides a kind of complex micro organism fungicide increase soil fertility, and mention
Preparation method and application are supplied.The complex micro organism fungicide passes through the collaboration facilitation of multiple-microorganism, can be effective
The content for improving available phosphorus in soil significantly improves soil fertility, increases crop yield, while can also inhibit the hair of pest and disease damage
Raw, application prospect is extensive.
One aspect of the present invention provides a kind of complex micro organism fungicide, includes bacillus subtilis (Bacillus
Subtilis), enterococcus faecalis (Enterococcus faecalis), bacillus coagulans (Bacillus coagulans) and
Trichoderma viride (Trichoderma viride).
The bacillus subtilis is that applicant screens the one plant of phosphate solubilizing bacteria obtained, is named as bacillus subtilis LLH-3
(Bacillus subtilis LLH-3) is preserved in the Chinese Typical Representative training of Wuhan, China Wuhan University on May 27th, 2019
Object collection is supported, deposit number is CCTCC NO:M2019402.
Each component and its weight ratio are respectively as follows: 60-80 parts of bacillus subtilis, excrement intestines ball in the complex micro organism fungicide
30-40 parts of bacterium, 20-40 parts of bacillus coagulans and 30-50 parts of Trichoderma viride.
It is further preferred that each component and its weight ratio are respectively as follows: bacillus subtilis in above-mentioned complex micro organism fungicide
80 parts, 30 parts of enterococcus faecalis, 30 parts of bacillus coagulans and 50 parts of Trichoderma viride.
It is further preferred that the bacterium numbering of the enterococcus faecalis is CGMCC 1.125.
It is further preferred that the bacterium numbering of the bacillus coagulans is CGMCC 1.4799.
It is further preferred that the bacterium numbering of the Trichoderma viride is CGMCC 3.3744.
Another aspect of the present invention provides the preparation method of above-mentioned complex micro organism fungicide, includes the following steps:
1) after activating bacillus subtilis, enterococcus faecalis, bacillus coagulans and Trichoderma viride respectively, expand culture extremely
Fermentation liquid is freeze-dried by logarithmic growth phase, and viable bacteria amount is made and is up to 1010-1011The hyperconcetration bacterium powder of CFU/g；
2) by hyperconcetration bacterium powder made from step (1) according to following weight ratio: 60-80 parts of bacillus subtilis, excrement intestines ball
30-40 parts of bacterium, 20-40 parts of bacillus coagulans and 30-50 parts of Trichoderma viride are configured to complex micro organism fungicide.
Above-mentioned bacillus subtilis, enterococcus faecalis, bacillus coagulans and Trichoderma viride distinguish preferred bacillus subtilis
CCTCC NO:M2019402, enterococcus faecalis CGMCC 1.125, Bacillus coagulans CGMCC 1.4799 and Trichoderma viride
CGMCC3.3744。
The present invention also provides application of the above-mentioned complex micro organism fungicide in crop planting.
The complex micro organism fungicide can be administered alone, and dosage is 20-80kg/ mus.
The complex micro organism fungicide can also be mixed in the ratio and inorganic fertilizer and/or organic fertilizer of 10-20% (mass ratio)
Application is closed, dosage is 30-100kg/ mus.
Beneficial effect
Complex micro organism fungicide provided by the invention passes through bacillus subtilis LLH-3, enterococcus faecalis, bacillus coagulans
With four kinds of bacterium collective effects of Trichoderma viride, collaboration facilitation effect can be generated, there is better fertilizer efficiency than single culture, produce
Unexpected technical effect.
The complex micro organism fungicide can effectively improve soil fertility, promote plant growth, fertilizer efficiency is significantly higher than identical
The inorganic fertilizer of amount of application.Compared with the control group, Chinese cabbage plant height in the experimental group of complex micro organism fungicide, ball height, transverse diameter point are applied
Do not improve 19.7%-21.0%, 10.6%-19.3%, 18.3%-22.2%, output increased 19.3-22.9%.It is described
The disease incidence of Seed detection and black spot can also be effectively reduced in complex micro organism fungicide, reduce respectively than inorganic fertilizer control group
45.8% and 67.2%, control efficiency is significant.
The complex micro organism fungicide can be administered alone, can also be in the ratio and inorganic fertilizer of 10-20% (mass ratio)
And/or organic fertilizer mixing application, so that crop yield is generally improved 30-50%, and environmentally friendly, is conducive to promote crops
Quality, push transformation of the traditional agriculture to the ecological agriculture, green agriculture, realize agricultural health, sustainable development.
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.Wherein:
Enterococcus faecalis is purchased from China General Microbiological culture presevation administrative center, and bacterium numbering is CGMCC 1.125；Condensation
Bacillus is purchased from China General Microbiological culture presevation administrative center, and bacterium numbering is CGMCC 1.4799；Trichoderma viride purchase
From China General Microbiological culture presevation administrative center, bacterium numbering is CGMCC 3.3744.
Embodiment 1 has the screening of phosphorus decomposing functional microorganism
1, pedotheque: the calacareous soil in Shandong Province, mountain area, the Qingzhou City west and south.
2, the preparation of soil dilution liquid: weigh 0.5g pedotheque be dissolved in be made in 4.5ml sterile water 1:10 soil it is molten
Then liquid is therefrom drawn the 0.5ml soil liquid and is placed in 4.5ml sterile water, the soil liquid of 1:100 is made, in this approach class
It pushes away, prepares 1:106-107Soil dilution solution.
0.1ml soil dilution solution is taken to be spread evenly across slightly solubility Phos solid medium (glucose 10g, (NH4)
2SO40.5g,NaCl 0.3g,KC1 0.3g,MgSO4·7H20 0.3g,FeSO4.7H20 0.03g,MnSO4.4H20
0.03g, Ca3 (PO4) 25.0g, distilled water 1000ml, pH 7.0-7.5, agar 20g, 115 DEG C of sterilizing 30min) on, 30 DEG C of trainings
It supports and is inverted culture 3 days in case, observe the bacterium colony grown on culture medium, become wherein there is 12 periphery of bacterial colonies to produce apparent color
Change, and have transparent circle generation, is respectively designated as LLH-1, LLH-2, LLH-3 ... ..., LLH-12.
The secondary screening of 2 phosphate solubilizing microorganism of embodiment
12 plants of bacterium that 1 primary dcreening operation of embodiment is obtained are inoculated into respectively on slightly solubility Phos solid medium, 30 DEG C of cultures 3
After it, the size of periphery of bacterial colonies transparent circle is observed, as a result, it has been found that the maximum three plants of bacterium of transparent circle are respectively LLH--2, LLH--3,
LLH--10。
The maximum three plants of bacterium of above-mentioned transparent circle are inoculated into 50mL slightly solubility Phos fluid nutrient medium (glucose respectively
10g,(NH4)2SO4 0.5g,NaCl 0.3g,KC1 0.3g,MgSO4.7H20 0.3g,FeSO4.7H20 0.03g,
MnSO4.4H20 0.03g, Ca3 (PO4) 25.0g, distilled water 1000ml, pH 7.0-7.5,115 DEG C of sterilizing 30min) in, 30
DEG C, 200rpm is cultivated 6 days, while the liquid phosphorus decomposing culture medium of any bacterium is not added as a control group；It detects in culture solution respectively
Number of viable.
The drafting of 2.1 phosphorus standard curves
Successively draw 5mg/l phosphorus standard solution 0.0,0.2,0.4,0.8,1.6,2.0,3.2,4.0ml in test tube, so
The anti-color developing agent 2ml of molybdenum antimony is respectively added afterwards, distilled water is settled to 20ml, shakes up and stands 20min, measures absorbance under 700nm wavelength.
The concentration of phosphorus is divided into each pipe at this time: 0.00,0.05,0.10,0.20,0.40,0.50,0.80,1.00mg/l.It is with phosphorus concentration
Abscissa, absorbance are ordinate, draw phosphorus standard curve.
The measurement of 2.2 culture solution available phosphorus contents
Culture solution each 5ml, the 8000rpm of above-mentioned three plants of bacterium are aseptically taken respectively, are centrifuged 5min, are taken supernatant,
It is diluted to suitable concentration, draws 0.5ml dilution into test tube, add 5ml distilled water, add 2 drops 2,4- dinitrophenol dinitrophenolate indicator,
Add the anti-color developing agent of 2ml molybdenum antimony, be then settled to 20ml with distilled water, shakes up and stand 20min, the colorimetric under 700nm wavelength, extinction
Angle value substitutes into the available phosphorus content in standard curve calculating supernatant, and concrete outcome is shown in Table 1.
The phosphorus decomposing effect of 1 different strains of table
It can be seen that three plants of bacterium that applicant screens from pedotheque from the data in table 1 and all have stronger solution
Indissoluble phosphorus (Ca3 (PO4) 2) in culture medium can be resolved into soluble available phosphorus, and significant effect by phosphorus ability.Wherein,
The phosphorus decomposing efficiency of LLH-3 bacterial strain is up to 85%, and available phosphorus content is up to 856mg/L, achieves unexpected effect.
The identification of 3 LLH-3 bacterial strain of embodiment
1) colonial morphology of LLH-3 bacterial strain:
The bacterium colony of the LLH-3 bacterial strain is flat, and rough surface is opaque, roughening after edge initial stage is smooth, and bacterium colony is presented
White is to dirty white, and gemma size is about 0.5-0.9 × 0.8-1.3 μm, ellipse or column, middle life or close middle raw；Gram
Reacting positive, catalase reaction is positive, V.P reacting positive, and Starch Hydrolysis test is positive, and indole test is positive, available
Glucose, arabinose, xylose and mannitol, 20-45 DEG C of growth temperature range, pH range 5-10.
2) molecular biology identification of LLH-3 bacterial strain:
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, and blast comparison is carried out in GenBank nucleic acid database.
SEQ ID NO:1 is as follows:
In conjunction with the colonial morphology and 16srDNA comparison result of LLH-3 bacterial strain, applicant determined that LLH-3 bacterial strain is withered grass bud
Spore bacillus (Bacillus subtilis) is named as bacillus subtilis LLH-3 (Bacillus subtilis LLH-3).
Applicant is on May 27th, 2019 by above-mentioned bacillus subtilis LLH-3 (Bacillus subtilis LLH-
3) it is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2019402.
The producing enzyme vitality test of 4 bacillus subtilis LLH-3 of embodiment
1, the preparation of bacterium solution
Choose the fresh bacillus subtilis LLH-3 bacterium mud of a ring, be inoculated in 100mL LB culture medium (1000ml water, 10.0
Peptone, 5.0 yeast extracts, 5.0gNaCl, pH7.0-7.2) in, 30 DEG C, 200rpm culture 12-16h seed liquor；It will kind
Sub- liquid is seeded in bactericidal nurishing broth bouillon with 5% inoculum concentration, 30 DEG C, 200rpm culture 28-32h, microscopy gemma yield
Stop fermented and cultured at 95% or more, obtaining viable bacteria content is 108-109The fermentation liquid of cfu/ml.
2, Enzyme activity assay
By fermentation liquid under the conditions of 4 DEG C, 8000rpm is centrifuged 5min, takes supernatant, by the following method measurement fermentation respectively
Phytase and cellulose enzyme activity in supernatant.The results show that the present invention, which screens the bacillus subtilis LLH-3 obtained, to be had
Stronger enzymatic productivity, phytase activity is up to 13.5U/mL in the fermented supernatant fluid, and cellulose enzyme activity is up to 4.04U/
ML, unexpected technical results have been achieved.
(1) phytase activity measuring method
The definition of enzyme-activity unit: under conditions of 30 DEG C, pH value are 5.0, the sodium phytate for being per minute 5mg/ml from concentration
It is an enzyme activity unit U that enzyme amount required for 1 μm of ol Phos is discharged in solution.
Measuring method: taking the sodium phytate solution that 4ml concentration is 7.5mmol/L, (pH5.0 0.25mol/L acetate buffer is matched
System), it is added in colorimetric cylinder, 30 DEG C of balance 5min add 2ml and suitably dilute through pH5.0 0.25mol/L acetate buffer
And the phytase enzyme solution balanced through 30 DEG C, it mixes in 30 DEG C of accurate insulation reaction 30min.After reaction, 4ml is added to terminate
Liquid (2 parts of nitric acid solutions (nitric acid: water=1:2), 1 part of 100g/L ammonium molybdate solution, 1 part of 2.35g/L Ammonium Vanadate Solution), mix with
Terminate reaction.Then it is placed at room temperature for 10min colour developing, measures light absorption value at spectrophotometer 415nm.
Enzyme activity calculation formula:
U=(A-A0-0.0016)×F/(0.0415×30)
In formula: A is the light absorption value of sample；A0For the light absorption value of blank sample；F is total dilution times before the reaction of practical sample liquid
Number；30 be enzyme digestion reaction time, min.
(2) Cellulase Activity Measurement Methods
The definition of enzyme-activity unit: under conditions of 50 DEG C, pH value are 6.0, the methylol for being per minute 5mg/ml from concentration
Enzyme amount required for 1 μm of ol reduced sugar of degradation release is an enzyme activity unit U in sodium cellulosate solution, and reduced sugar is with glucose
Equivalent.
Measuring method: taking three test tubes that 0.5mL CMC substrate is respectively added, 50 DEG C of water-bath preheatings together with enzyme solution to be measured
5min.0.5mL prepare liquid, and timing are respectively added in the first and second test tube, reacts 15min in 50 DEG C of water-baths.Three after having reacted
1.5mL DNS reagent is respectively added in branch test tube, and always adds the enzyme solution to be measured of 0.5mL in third branch test tube.It takes out and shakes up three
After branch test tube, 5min is reacted in boiling water bath.It is rapidly cooled to room temperature, it is fixed to 5.0mL with water.It is pair with third branch test tube test solution
The absorbance that the first and second test tube test solution is surveyed under 540nm wavelength condition is impinged upon, absorbance is advisable between 0.25-0.35.Enzyme to be measured
The absolute value of the difference of the absorbance of liquid reaction solution and horizontal control enzyme solution reaction solution absorbance is no more than 0.015.
Enzyme activity X=(magnitudes/180/15/0.5 such as glucose) × n
Wherein: X --- enzyme activity unit, IU/g (mL)；
180 --- glucose is converted into micromole from microgram；
15 --- the reaction time of prepare liquid and substrate；
0.5 --- the enzyme liquid amount to be measured of reaction is added；
N --- extension rate.
Application of the 5 bacillus subtilis LLH-3 of embodiment in the experiment of spinach field production
1, place: Qingdao City Laixi spinach planting greenhouse, soil integral status are more uniform.
2, experimentation
30 test blocks are set, and each test block is the square region of 2m × 2m, and 1 meter is kept between each test block
Interval.
Experiment sets 3 groups altogether: 1. blank control group: not adding any substance；2. medium treatment group: in each test block
In press 40mL/m2Ratio uniform spray the nutrient broth medium of above-mentioned sterilizing, then by the soil of surface layer 5-10cm thickness into
Row effectively mixes；3. bacillus subtilis LLH-3 processing group: pressing 60mL/m in each test block2Ratio uniform spray it is real
Apply bacillus subtilis LLH-3 zymocyte liquid (10 described in example 48-109Cfu/ml), then the soil of surface layer 5-10cm thickness is carried out
Effectively mix.10 test blocks of each group of random selection.
1) seed treatment: with 5% sodium hypochlorite surface sterilizing spinach seed 10min, wash with distilled water 3-4 removing
30min natural drying is placed after sodium hypochlorite at normal temperature.
2) sowing and harvest: 25g spinach seed, regular watering and management are uniformly sowed in each test block, does not apply fertilizer
Material.After sowing 50 days, whole spinach is harvested, and detect the fresh weight and dry weight of each test block spinach respectively, calculate each processing
The mean fresh and average dry weight of group spinach, are compared.
3) while harvesting spinach, the soil sample of each test block is acquired respectively, using available phosphorus in Olsen method detection soil sample
Content, and be compared.
Test result shows: compared with blank control group, the mean fresh and dry weight of medium treatment group spinach mention respectively
High by 4.8% and 4.1%, available phosphorus content improves 3.2%；The mean fresh of bacillus subtilis LLH-3 processing group spinach
295.7% and 228.2% has been respectively increased than blank control group with dry weight, 290.9% He is respectively increased than medium treatment group
224.1%, available phosphorus content improves 78.5% than space management group, improves 75.3% than medium treatment group.
The above results show that the bacillus subtilis LLH-3 that the present invention screens extremely significant can increase soil fertility, thus
The yield for greatly improving long-term cropping can be used as bio-fertilizer and be widely used in agricultural production.
Application of the 6 bacillus subtilis LLH-3 of embodiment in seedling cultivation of rice
1, prepared by bacterium powder
The fresh bacillus subtilis LLH-3 bacterium mud of a ring is chosen, is inoculated in 100mL LB culture medium, 30 DEG C, 200rpm
Cultivate 12-16h seed liquor；Seed liquor is seeded in bactericidal nurishing broth bouillon with 5% inoculum concentration, 30 DEG C,
200rpm cultivates 28-32h, and microscopy gemma yield stops fermented and cultured at 95% or more；By fermentation liquid centrifugation (8000rpm,
Thallus 5min) is collected, dextrin is added and stirs and evenly mixs, vacuum freeze drying, it is 10 that bacterium amount, which is made,9-1010The withered grass gemma of CFU/g
Bacillus LLH-3 bacterium powder.
2, experimentation
(1) place: the seedling raising greenhouse of Dandong City, Liaoning Province is tested.
(2) experimental design:
1. blank control group: only with seedling medium (strengthening development in agricultural science and technology Co., Ltd purchased from Harbin Miao Sheng)；
2. bacillus subtilis CGMCC1.8886 control group: 1.5kg bacillus subtilis CGMCC1.8886 bacterium powder (109-
1010CFU/g it) is mixed with 1 ton of seedling medium even；
3. bacillus subtilis LLH-3 processing group: 1.5kg bacillus subtilis LLH-3 bacterium powder (109-1010CFU/g) with 1
Ton seedling medium is mixed even；
The management such as sowing, irrigation and fertilising of two above processing group is identical, and entire nursery stage is about 40 days.
3, experimental result and analysis
(1) rice seedling form and chlorophyll content evaluation
100 plants of rice shoots are extracted from the central randomization of each processing group seedlings nursing plate, carry out examining seedling after being rinsed well with clear water, and examine
Survey the chlorophyll content of the second leaf.It the results are shown in Table 3.
The influence that 2 bacillus subtilis LLH-3 of table grows rice seedlings strain
From the data of table 2 it is found that compared with blank control group, bacillus subtilis LLH-3 processing group provided by the invention
Rice seedling plant height, stem thickness, radical, leaf age, the second leaf is long, and chlorophyll content has been respectively increased 24.1%, 27.3%,
29.3%, 30.9%, 10.3%, 10.2%, effect highly significant.And use commercially available bacillus subtilis CGMCC1.8886
Treated rice seedling, only plant height and stem thickness have been respectively increased 10.2% and 8.9%, and radical, leaf age, the second leaf be long and leaf
Chlorophyll contents increase unobvious.To illustrate, bacillus subtilis LLH-3 provided by the invention can significantly improve educating for rice
Seedling quality, effect are substantially better than commercially available bacillus subtilis.
(2) Seedling Quality in Rice is evaluated
Hundred plant dry weights and seedling vigorous index are the important indicators of seedling quality height.
Seedling vigorous index=(stem thickness × 100 plant rice shoot complete stool dry weight)/plant height.
Hundred plant dry weight of rice seedling and nursery index of bacillus subtilis LLH-3 processing group provided by the invention compare blank
Control group has increased separately 38.56% and 58.07%, increases separately than bacillus subtilis CGMCC1.8886 control group
30.48% and 48.76%, unexpected technical results have been achieved.
The measurement of 7 bacillus subtilis LLH-3 bacteriostasis of embodiment
1, pathomycete is cultivated:
In aseptic operating platform, peanut root rot bacterium, the southern blight of the fritter of about 0.5cm × 0.5cm are taken respectively with tweezers
Bacterium, Streptomyces scabies and brown patch germ are seeded in PDA culture medium, and 28 DEG C of inversions are cultivated 3 days.
2, inoculation face-off bacterium:
It is long to when accounting for about culture dish 1/3 when pathomycete, it is being inoculated with bacillus subtilis LLH- respectively at fungi 2cm
3, the fungi not to be inoculated with bacillus subtilis LLH-3 measures after continuation is cultivated 3 days under the conditions of 28 DEG C respectively as control
The colony radius of each pathomycete calculates bacteriostasis rate.
Bacteriostasis rate=[(control fungi growth radius-processing fungi grows radius)/control fungi grows radius] ×
100%.
The results show that bacillus subtilis LLH-3 has apparent antagonism to above-mentioned four pathomycetes of cultivating peanut.Its
In, LLH-3 is most strong to the inhibiting effect of peanut root rot bacterium and Streptomyces scabies, and inhibiting rate is up to 87.4% and 81.6% respectively；
Relatively weak to the inhibiting effect of Sclerotium rolfssi and brown spot, inhibiting rate is respectively 63.3% and 59.8%.
Application of the 8 bacillus subtilis LLH-3 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) bacillus subtilis CGMCC1.8886 processing group: respectively in sowing time, after planting 15d, 30d, 45d be using withered
Careless bacillus LLH-3 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 30mL of dilution every time；
(4) bacillus subtilis LLH-3 processing group: respectively sowing time, after planting 15d, 30d, 45d utilize withered grass gemma
Bacillus LLH-3 fermentation liquid (108-109Cfu/ml) pouring root, every plant is poured 100 times of fermentation liquid about 30mL 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 bacillus subtilis LLH-3 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 more than to the prevention and treatment efficiency of root rot and shot hole
80%, the prevention and treatment efficiency to southern blight and brown spot is more than 60%, is significantly higher than the control efficiency of chemicals treatment group carbendazim.And
Commercially available bacillus subtilis CGMCC1.8886 has certain prevention and treatment to peanut root rot, southern blight, shot hole and brown spot
Effect, but preventing and treating efficiency is only 11.7%-13.6%, is far below bacillus subtilis LLH-3 of the present invention.
The above results show that bacillus subtilis LLH-3 provided by the invention wants the control efficiency of peanut Common Diseases
It is significantly better than traditional chemical bactericide, and environmentally friendly, is conducive to the quality for promoting crops, can be widely applied to green
In agricultural production.
Embodiment 9
A kind of complex micro organism fungicide, each component and its weight are respectively as follows: 60 parts of bacillus subtilis LLH-3, excrement intestines
40 parts of coccus, 40 parts of bacillus coagulans and 45 parts of Trichoderma viride.
The preparation method of above-mentioned complex micro organism fungicide, includes the following steps:
1) after activating bacillus subtilis LLH-3, enterococcus faecalis, bacillus coagulans and Trichoderma viride respectively, expand
Fermentation liquid is freeze-dried to logarithmic growth phase, viable bacteria amount is made and is up to 10 by culture10-1011The hyperconcetration bacterium powder of CFU/g；
2) by hyperconcetration bacterium powder made from step (1) according to following weight ratio: 60 parts of bacillus subtilis LLH-3, excrement intestines
40 parts of coccus, 40 parts of bacillus coagulans and 45 parts of Trichoderma viride are configured to complex micro organism fungicide.
Embodiment 10
A kind of complex micro organism fungicide, each component and its weight ratio are respectively as follows: 70 parts of bacillus subtilis LLH-3, excrement
33 parts of enterococcus, 20 parts of bacillus coagulans and 30 parts of Trichoderma viride.
Preparation method is referring to embodiment 9.
Embodiment 11
A kind of complex micro organism fungicide, each component and its weight ratio are respectively as follows: 80 parts of bacillus subtilis LLH-3, excrement
30 parts of enterococcus, 30 parts of bacillus coagulans and 50 parts of Trichoderma viride.
Preparation method is referring to embodiment 9.
Application of 12 complex micro organism fungicide of embodiment in Chinese cabbage is planted
1, influence of the complex micro organism fungicide to Chinese cabbage growth performance and yield
It chooses that soil property is fertile, the uniform plot of soil fertility in Qingdao City Laixi vegetables flake, transplants Chinese cabbage seedling, respectively at
The complex micro organism fungicide that the embodiment of the present invention 9,10,11 is prepared, dose 10kg/ are applied at 1 month, two months respectively
Mu, meanwhile, to apply equivalent inorganic fertilizer as a control group, 3 months whens, test and assess to the Chinese cabbage of experimental group and control group, as a result
As shown in table 7.
Influence of 7 complex micro organism fungicide of table to Chinese cabbage growth performance and yield
| Processing | Fertilising | Plant height (cm) | Ball height (cm) | Transverse diameter (cm) | Per mu yield (kg) |
| Control group | Inorganic fertilizer | 30.5 | 21.8 | 15.3 | 3126 |
| Experimental group 1 | Embodiment 9 | 36.9 | 24.1 | 18.1 | 3728 |
| Experimental group 2 | Embodiment 10 | 36.5 | 24.5 | 18.4 | 3760 |
| Experimental group 3 | Embodiment 11 | 36.9 | 26.0 | 18.7 | 3842 |
It can be seen that compared with the control group from the data in table 7, apply Chinese cabbage strain in the experimental group of complex micro organism fungicide
19.7%-21.0%, 10.6%-19.3%, 18.3%-22.2%, output increased has been respectively increased in high, ball height, transverse diameter
19.3-22.9%, obvious effect of increasing production.To illustrate, complex micro organism fungicide produced by the invention can effectively improve soil fertilizer
Power, promotes Chinese Cabbage, and fertilizer efficiency is significantly higher than the inorganic fertilizer of identical amount of application.
Moreover, applying each finger of Chinese cabbage of the experimental group 3 of complex micro organism fungicide described in embodiment 11 in three experimental groups
Mark is apparently higher than experimental group 1 and experimental group 2.
2, the synergistic effect of four kinds of bacterium is evaluated in complex micro organism fungicide
(1) microbial bacterial agent
Sample 1: bacillus subtilis LLH-3 bacterium powder, living bacteria count about 1010cfu/g；
Sample 2: enterococcus faecium bacterium powder, living bacteria count about 1010cfu/g；
Sample 3: bacillus coagulans bacterium powder, living bacteria count about 1010cfu/g；
Sample 4: trichoderma viride powder, living bacteria count about 1010cfu/g；
Sample 5: complex micro organism fungicide described in embodiment 11, by bacillus subtilis LLH-3, enterococcus faecalis, condensation bud
Spore bacillus and Trichoderma viride composition, total living bacteria count about 1010cfu/g；
(2) experimentation
It chooses that soil property is fertile, the uniform plot of soil fertility in Qingdao City Laixi vegetables flake, transplants Chinese cabbage seedling, respectively at
Apply microbial bacterial agent described in sample 1-5 at 1 month, two months respectively, 10kg/ mus of dose, meanwhile, it is inorganic to apply equivalent
As a control group, 3 months whens, test and assess to the Chinese cabbage of experimental group and control group to fertilizer, and the results are shown in Table 8.
Influence of 8 complex micro organism fungicide of table to Chinese cabbage growth performance and yield
| Processing | Fertilising | Ball height (cm) | Transverse diameter (cm) | Per mu yield (kg) | Per mu yield is compared with CK ± % |
| Control group | Inorganic fertilizer | 21.8 | 15.3 | 3126 | - |
| Experimental group 1 | Sample 1 | 24.7 | 17.5 | 3620 | 15.8% |
| Experimental group 2 | Sample 2 | 17.1 | 14.2 | 2428 | - 22.3% |
| Experimental group 3 | Sample 3 | 22.8 | 16.3 | 3405 | 8.9% |
| Experimental group 4 | Sample 4 | 22.0 | 15.4 | 3186 | 1.9% |
| Experimental group 5 | Sample 5 | 26.0 | 18.7 | 3842 | 22.9% |
From the data of table 8 it can be seen that
(1) experimental group 1 of bacillus subtilis LLH-3 bacterium powder is administered alone and bacillus coagulans bacterium powder is administered alone
The growth performance of 3 Chinese cabbage of experimental group is significantly higher than inorganic fertilizer control group, and 15.8% and 8.9% is respectively increased than control group in per mu yield,
Illustrate bacillus subtilis LLH-3 or bacillus coagulans independent role, can effectively improve soil fertility, effect is better than equivalent
Inorganic fertilizer；
(2) growth performance and per mu yield that 2 Chinese cabbage of experimental group of enterococcus faecium bacterium powder is administered alone are below inorganic fertilizer control
Group illustrates that enterococcus faecium independent role is not so good as the inorganic fertilizer of equivalent to the promotion effect of soil fertility；
(3) growth performance and per mu yield and inorganic fertilizer control group base of 4 Chinese cabbage of experimental group of trichoderma viride powder is administered alone
This quite illustrates that Trichoderma viride independent role is suitable with two inorganic fertilizers are waited to the promotion effect of soil fertility.
(4) application equivalent is made of bacillus subtilis LLH-3, enterococcus faecalis, bacillus coagulans and Trichoderma viride
The experimental group 5 of complex micro organism fungicide, the growth performance of Chinese cabbage are significantly higher than experimental group 1 and experimental group 3, and per mu yield is mentioned than control group
High 22.9%, also significantly greater than experimental group 1 and experimental group 3.
The above results explanation, bacillus subtilis LLH-3 in complex micro organism fungicide provided by the invention, enterococcus faecalis,
Four kinds of bacterium collective effects of bacillus coagulans and Trichoderma viride, can generate collaboration facilitation effect, have than single culture more preferable
Fertilizer efficiency, produce unexpected technical effect.
In addition, applicant also found during the experiment, complex micro organism fungicide provided by the invention is in addition to can substantially mention
High soil fertility promotes except plant growth, moreover it is possible to the disease incidence of Seed detection and black spot be effectively reduced, compare inorganic fertilizer
Control group reduces 45.8% and 67.2%, significant effect respectively.
Application of 12 complex micro organism fungicide of embodiment in corn planting
1, test place: the village Pingdu Li Jiatun, Qingdao City, soil integral status are more uniform.
2, laboratory sample:
1) sample: complex micro organism fungicide described in embodiment 11, total living bacteria count about 1010cfu/g。
2) conventional composite fertilizer sample: pacify compound fertilizer for Stanley three, wherein N-P2O5-K2O, 26-14-14, total nutrient >=
54%.
3, experimentation:
1) for trying soil pH value 6.7-7.0, after measured, available phosphorus content is about 1.53mg/kg in soil.
2) corn variety: first jade 335.
3) each 400 square meter of hillslope processes area, totally 5 processing, each processing set 3 in parallel, and each hillslope processes are random
Arrangement.
Control group: only application conventional composite fertilizer, amount of application are 40kg/ mus；
Processing group 1: complex micro organism fungicide provided by the invention is multiple to tradition by the adding proportion of 10% (mass ratio)
Hefei is coated, and amount of application is 40kg/ mus；
Processing group 2: complex micro organism fungicide provided by the invention is multiple to tradition by the adding proportion of 10% (mass ratio)
Hefei is coated, and amount of application is 30kg/ mus.
Processing group 3: complex micro organism fungicide provided by the invention is multiple to tradition by the adding proportion of 10% (mass ratio)
Hefei is coated, and amount of application is 25kg/ mus.
Processing group 4: complex micro organism fungicide provided by the invention is multiple to tradition by the adding proportion of 15% (mass ratio)
Hefei is coated, and amount of application is 20kg/ mus.
Corn is sowed simultaneously in seed manure on May 11st, 2018,50 ± 1cm of line-spacing, 25 ± 1cm of spacing in the rows.Other fields are daily
Each hillslope processes are managed to be consistent.Each hillslope processes corn was harvested on September 25th, 2019 respectively, measures yield, and calculate
The volume increase situation of each processing group corn, concrete outcome are shown in Table 9.
3, experimental result
The Yield comparison of 9 different disposal group corn of table
From the result of table 9 it is recognised that compared with the control group, in the case where identical dose, adding the present invention and providing
1 corn yield of processing group of complex micro organism fungicide improve 30.7%；When dose reduces 25%, composite microbial is added
The processing group 2 of object microbial inoculum is still able to achieve corn yield increasing 22.5%；When dose reduces 50%, complex micro organism fungicide is added
3 corn yield of processing group remain to it is more slightly higher than control group, realize weight-reducing the not underproduction, achieve unexpected effect.Moreover, with
The increase of complex micro organism fungicide adding proportion (increasing to 15% by 10%), corn yield and 3 phase of processing group of processing group 4
Than being significantly improved.
The above results show that complex micro organism fungicide provided by the present invention passes through bacillus subtilis LLH-3, excrement intestines ball
Collaboration facilitation between bacterium, bacillus coagulans and Trichoderma viride, can effectively improve the content of available phosphorus in soil, not only
Crop yield can be greatly improved, reduction environmental pollution is additionally aided.
The complex micro organism fungicide can be administered alone, can also be in the ratio and inorganic fertilizer of 10-20% (mass ratio)
And/or organic fertilizer mixing application, crop yield can be made to improve 30-50%, wide market.
Sequence table
<110>Qingdao force favour biotech inc
<120>a kind of complex micro organism fungicide increase soil fertility and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 960
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 1
ctctagggtt ttcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa 60
accacatgct ccaccgcttt tgcgggcctc cgtcaattcc tttgagtttt agccttgcgg 120
ccgtactccc caggcggagt ggttaatgcg ttaacttcag cactaaaggg cggaaaccct 180
ctaacactta gaactcatcc tttacggcgt ggactaccag ggtatctaat cctgtttgct 240
ccccacgctt tcgcgcctca gtgtcagtta cagaccagaa agtcgccttc gccactggtg 300
ttcctccata tctctacgca tttcaccgct acacatggaa ttccactttc ctcttctgca 360
ctcaagtctc ccagtttcca atgaccctcc acggttgagc cgtgggcttt cacatcagac 420
ttaagaaacc acctgcgcgc gctttacgcc caataattcc ggataacgct tgccacctac 480
gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaggt accgtcaagg 540
tgccagctta ttcaactagc acttgttctt ccctaacaac agagttttac gacccgaaag 600
ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcggg ggattccgtc 660
ctgctgcctc ccctaggagg ctgggccgtg tctcagtccc agtgtggccg atcaccctct 720
caggtcggct acgcatcgtt gccttggtga gccgttacct caccaactag ctaatgcgac 780
gcgggtccat ccataagtga cagccgaagc cgcctttcaa tttcgaacca tgcagttcaa 840
aatgttatcc ggtattagcc ccggtttccc ggagttaccc cagtcttatg ggcaggttac 900
ccacgtgtta ctcacccgtc cgccgctaac tcactcgagc atgctactag cttttgcccc 960
Claims (9)
1. a kind of complex micro organism fungicide, which is characterized in that the complex micro organism fungicide includes bacillus subtilis
(Bacillus subtilis), enterococcus faecalis (Enterococcus faecalis), bacillus coagulans (Bacillus
) and Trichoderma viride (Trichoderma viride) coagulans.
2. complex micro organism fungicide as described in claim 1, which is characterized in that the deposit number of the bacillus subtilis is
CCTCC NO:M2019402.
3. complex micro organism fungicide as claimed in claim 1 or 2, which is characterized in that each in the complex micro organism fungicide
Component and its weight ratio are respectively as follows: 60-80 parts of bacillus subtilis, 30-40 parts of enterococcus faecalis, 20-40 parts of bacillus coagulans
With 30-50 parts of Trichoderma viride.
4. complex micro organism fungicide as claimed in claim 3, which is characterized in that each component in the complex micro organism fungicide
And its weight ratio is respectively as follows: 80 parts of bacillus subtilis, 30 parts of enterococcus faecalis, 30 parts of bacillus coagulans and Trichoderma viride 50
Part.
5. the complex micro organism fungicide as described in claim 1-4 is any, which is characterized in that the strain of the enterococcus faecalis is compiled
It number is CGMCC 1.125, the bacterium numberings of the bacillus coagulans is CGMCC 1.4799, the bacterium of the Trichoderma viride
Kind number is CGMCC 3.3744.
6. the preparation method of any complex micro organism fungicide of claim 1-5, which is characterized in that the method includes such as
Lower step:
(1) after activating bacillus subtilis, enterococcus faecalis, bacillus coagulans and Trichoderma viride respectively, expand and cultivate to right
In number growth period, fermentation liquid is freeze-dried, viable bacteria amount is made and is up to 1010-1011The hyperconcetration bacterium powder of CFU/g；
(2) by hyperconcetration bacterium powder made from step (1) according to following weight ratio: 60-80 parts of bacillus subtilis, enterococcus faecalis
30-40 parts, 20-40 parts of bacillus coagulans and 30-50 parts of Trichoderma viride are configured to complex micro organism fungicide.
7. application of any complex micro organism fungicide of claim 1-5 in crop planting.
8. the use as claimed in claim 7, which is characterized in that the complex micro organism fungicide can be administered alone, and dosage is
20-80kg/ mus.
9. the use as claimed in claim 7, which is characterized in that the complex micro organism fungicide can be by 10-20%(mass ratio)
Ratio mix application with inorganic fertilizer and/or organic fertilizer, dosage is 30-100kg/ mus.
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