CN107142210A - A kind of compound method of hard stalk fermentation microbial inoculum - Google Patents
A kind of compound method of hard stalk fermentation microbial inoculum Download PDFInfo
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- CN107142210A CN107142210A CN201710268176.9A CN201710268176A CN107142210A CN 107142210 A CN107142210 A CN 107142210A CN 201710268176 A CN201710268176 A CN 201710268176A CN 107142210 A CN107142210 A CN 107142210A
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- bacillus subtilis
- whiterot fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a kind of compound method of hard stalk fermentation microbial inoculum, comprise the following steps:The first step, selectes strain;The culture and bacteria B. subtilis culture of second step, the culture of strain, including fungi whiterot fungi;3rd step, takes the whiterot fungi after expansion culture well mixed according to 2: 3 quality proportioning with the bacillus subtilis after expansion culture, the container of the prior disinfection of loading, obtained microbial inoculum of the invention.The mix bacterium agent prepared using the inventive method, for hard stalk fermentation microbial inoculum, can effectively improve the fermenting speed of hard stalk, so as to be agricultural production service for manufacturing organic fertilizer.The recycling of stalk suitable for agricultural production.
Description
Technical field
The present invention is relevant with microorganism, specifically, relevant with fermenting agent, furthermore, it is understood that being related to the fermentation of stalk
Microbial inoculum.
Technical background
The content of organic matter of crop material is high, and rich in the nutrient needed for crop growth, it is to have to be practically free of impurity
Machine fertilizer preferably raw material, has higher agricultural value by the product of its production.However, the phosphorus content of stalk is higher, C/N ratios
Value is high, is not appropriate for the raw material separately as organic fertilizer, and needs to coordinate with the excrement class that phosphorus content is relatively low, C/N ratios are low
Use.Lignin, cellulose, the hemicellulose level of general soft crop material are relatively low, are easier to become thoroughly decomposed, such as paddy rice, small
Wheat, rape, corn etc..And hard stalk, such as husk, the lignin of wood chip and bark, cellulose, hemicellulose level it is higher,
C/N is not easily decomposed more than 100, and degradation time is long, can the original form of longer-term holding.But soya bean, cotton, tobacco etc.
Bar, generally as the sludge, the auxiliary material of Animal fecal pollution that moisture is high, C/N ratios are low, it is necessary to control consumption, material particular diameter will
Small, particle is thinner, could accelerate degradation speed.
For different types of stalk, it is accelerated degraded and be changed into fertilizer, traditional method is exactly by the way that stalk is sent out
Ferment.Two Main changes can occur for organic fertilizer fermentation process, and one is complicated organic substance (cellulose, hemicellulose, egg
White matter etc.) simple inorganic compound is decomposed into, this is the process of nutrient validation, referred to as mineralization process；Another is
The intermediate product formed during organic matter is mineralization, then synthesize than original increasingly complex humus, referred to as humification
Process.It is well known that fermentation is the biochemical process of a microorganism, it is necessary to using bacterium.The stalk of different hardness, the bacterium used
Plant also different.
Through retrieval, it is related to zymophyte in Chinese patent database or the patent application of microbial inoculum is not a lot, what is retrieved has
No. ZL2006101461458《The method of bolt bacterium AH28-2 preparing laccase by solid-state fermentation》, No. 2011104132326《A kind of large intestine
Escherichia RB3 bacterial strains and the method that acetylesterase is prepared with its liquid state fermentation》, No. 2014100753519《A kind of watermelon is used anti-
Sick high yield EM bacteria agent fermentation composite fertilizer》, No. 2014100752658《A kind of growth of watermelon EM bacteria agent fermentation composite fertilizer》、
No. 2015103288138《A kind of pigeon dung EM bacterium fermentation methods》Deng these fermenting agents can not be used for the fermentation of stalk.It is applied to
The patent application of stalk fermentation has No. 2011102578402《A kind of straw fermentation complex microbial inoculant and its application》, it is by withered grass
Bacillus, trichoderma aureoviride, saccharomyces cerevisiae, aspergillus oryzae, expansion mould, Trichoderma viride are composited.It there is no at present for hard
The patent application of stalk fermentation microbial inoculum.
The content of the invention
The present invention is intended to provide a kind of compound method of hard stalk fermentation microbial inoculum, the fermentation speed to improve hard stalk
Degree, so as to be agricultural production service for manufacturing organic fertilizer.
The compound method for hard stalk fermentation microbial inoculum that inventor provides comprises the following steps：
The first step, selectes strain：Sent out using fungi whiterot fungi and bacteria B. subtilis as hard stalk is formulated for
The raw material strain of yeast-like fungi agent；The fungi whiterot fungi is purchased from China General Microbiological culture presevation administrative center (China
General Microbiological Culture Collection Center, CGMCC), fungi whiterot fungi drops to cellulose
Solution ability is stronger；What the bacteria B. subtilis was voluntarily screened and cultivated.
For the raw material strain of selection, it is required for by conventional means, determines it and tobacco the main pathogenic fungi antagonism is made
With the active bacterial strain molecule that identification is selected；Determine its feasibility for being used to prepare mix bacterium agent.
Second step, the culture of strain：
(1) culture of fungi whiterot fungi
1. the culture of fungi whiterot fungi parent species
Iblet is soaked in advance and packed after 72h, and it is standby in being cooled down after high pressure steam sterilization 30-40min at 121 DEG C；
Then the whiterot fungi after activation is inoculated in maize culture medium, puts in constant incubator 28 DEG C of cultures, when mycelia cover with it is whole
It is standby after individual iblet；
2. the expansion culture of fungi whiterot fungi
Cultured whiterot fungi parent species are linked into wheat bran cotton seed hulls mixed culture medium, 28 DEG C of trainings in constant incubator are put
Support, after media surface covers with mycelia, throwing 1 time is turned over per 48h, until whiterot fungi is covered with whole wheat bran cotton seed hulls culture medium.
(2) bacteria B. subtilis culture
1. the preparation of bacillus subtilis seed liquor：By the bacillus subtilis strain of preservation in NA solid mediums, flat
16h is cultivated on plate in 37 DEG C of incubators, then picking single bacterium is fallen within seed culture medium, 37 DEG C, 170rmin-1Under the conditions of
Culture 16h is used as seed liquor.
2. the solid fermentation of bacillus subtilis：Wheat bran and the cotton seed hulls mixture according to the composition of quality proportioning 3: 1 are existed
121 DEG C, 30min intervals sterilizing twice, obtain wheat bran cotton seed hulls mixed culture medium；Then bacillus subtilis seed liquor is pressed
10% inoculum concentration is linked into wheat bran cotton seed hulls mixed culture medium, and the addition of wheat bran cotton seed hulls mixed culture medium is 20%,
Water content is 60%, and fermentation temperature is 28 DEG C, and it is 1 time/24h to turn over throwing number of times；Realize the solid fermentation of bacillus subtilis.
3rd step whiterot fungi, the preparation of bacillus subtilis mix bacterium agent
Take second step to expand the whiterot fungi after culture and expand the bacillus subtilis after culture according to 1: 3 quality proportioning
It is well mixed, then load the container of prior disinfection according to every bag 1000g amount, be stored in cool place, dry, divulge information, keeping away
At light, avoid and poisonous substance together accumulating.18 months shelf-lifves under the conditions of above-mentioned accumulating.
Determined described in the above method first step to tobacco the main pathogenic fungi antagonism, be to tobacco black shank bacterium
(Phytophthora parasitica var.nicotiana), tobacco ralstonia solanacearum (Pseudomonas
Solanacearum antagonistic effect) is carried out；It is described identification select active bacterial strain molecule include PCR amplification, determined dna sequence with
Analysis.
Mixed culture medium described in above method second step is wheat bran cotton seed hulls and cotton seed hulls according to the mixed of quality proportioning 3: 1
Compound, sterilizes obtained twice at 121 DEG C, 30min intervals；The NA solid mediums be with beef extract 3.0g, NaCl5.0g,
Obtained by peptone 10.0g, agar 15-20g, water 1000mL are prepared, its pH7.2-7.5；The seed culture medium is to use beef
Obtained by cream 3.0g, NaCl5.0g, peptone 10.0g, water 1000mL are prepared, its pH7.2-7.5.
Container described in the step of the above method the 3rd can be one kind in packaging bag, pail pack, packing jar, vial.
In order to be formulated for hard stalk fermentation microbial inoculum, inventor has carried out following research and experiment：
Separation, the purifying of 1 antagonistic bacterium
The dilution of 1.1 samples weighs each 5g of tobacco rhizosphere pedotheque, organic fertilizer (being accurate to 0.01g), is separately added into band
In the 45mL sterilized waters of bead, stand 20min, 200r/min fully vibrates 30min on rotary shaker, sample it is female
Liquid.10 times of doubling dilutions are carried out to sample mother liquor.
1.2, which are loaded and cultivate each sample, takes 3 continuous suitable dilution factors, and bacterium takes 10-4、10-5、10-6.Inhale respectively
Different dilution factor sample suspension 0.1mL are taken, are added on solid NA culture medium flat plates well prepared in advance, coating is until liquid quilt
Fully absorb, each 3 repetitions of gradient.Bacterium is cultivated under the conditions of being positioned over 28 DEG C.
Purifying, the preservation of 1.3 separation of bacterial are purified isolated bacterium by plate streak, and will purifying
Thing is accessed on corresponding slant medium and cultivated, and 4 DEG C save backup.
Measure of 2 separation of bacterial to tobacco the main pathogenic fungi antagonism
2.1 indicate the selection of tobacco pathogen with tobacco black shank bacterium (Phytophthora parasitica
Var.nicotiana), tobacco ralstonia solanacearum (Pseudomonas solanacearum) utilizes flat board as target pathogens
Opposite culture method and inhibition zone method, Screening of Antibacterial Activities is carried out to isolated bacterium.
2.2 bacteriums will activate on PDA plate respectively to the antagonistic effect of tobacco black shank bacterium for examination tobacco disease fungus
After 3d, beaten with card punch (diameter 6mm) edge for examination tobacco pathogen colony edge and take bacterium dish standby, isolated bacterial strain is existed
Activated on NA culture medium flat plates after 24h, take a ring dominant strain to be rule in PDA plate center with oese, for examination tobacco cause of disease
Bacterium is seeded in PDA plate both sides respectively, and vaccination is away from plate center 2.5cm.Simultaneously using be only inoculated with the flat board of pathogen as pair
According to often processing sets 3 repetitions, is placed in incubated under dark condition in 25 DEG C of illumination boxs.When culture is to 3d, see day by day
Record pathogen colony radius are examined, according to more each isolated strains of mycelial growth inhibition rate to the suppression for trying tobacco disease fungus
Effect.
Colony radius=measurement bacterium colony mean radius -3.0mm
Mycelial growth inhibition rate (%)=(control colony radius-processing colony radius)/control colony radius × 100
Coating is seeded on flat board and cultivated after 24h after 2.3 bacteriums activate bacterium to the antagonistic effect of tobacco ralstonia solanacearum,
Bacterium dish aseptically is made with 6mm card punch, bacterium dish is positioned over into coating is inoculated with the flat board of tobacco ralstonia solanacearum, 28
DEG C culture, every 12 h observe 1 time, culture 48h after with crossing method determine inhibition zone diameter, often processing be repeated 3 times, with
Sterile culture medium punching replaces bacterium dish as control (CK).With respect to bacteriostasis rate (%)=(antibacterial circle diameter-bacterium dish diameter)/bacterium
Dish diameter × 100%.
Antagonistic effect result of 2.4 bacteriums to tobacco pathogen
Obtain 1 plant by flat board opposite culture and inhibition zone method screening and have to tobacco ralstonia solanacearum, tobacco black shank bacterium
The bacterial strain of strong antagonism, for next step identification.
The Molecular Identification of 3 active bacterial strains
3.1PCR amplification
Extracting the genomic DNA with obvious bacteriostatic activity bacterium bacterial strain using RNA isolation kit, (kit is given birth to purchased from Shanghai
Work bioengineering Co., Ltd).Primer used in amplification and sequencing is XJF:5’-AGA GTT TGA TCA TGG CTC AG-
3 ' and XJR:5 '-AAG GAG GTG ATC CAG CCG CA-3 ', by Shanghai, Sheng Gong bioengineering Co., Ltd synthesizes.PCR is anti-
20 μ L reaction systems, including template DNA solution (concentration is 50ng/ μ L) 1 μ L, 10 × Buffer 2.0 μ L, dNTP should be used
(concentration 2.5mM) 2.0 μ L, primer XJF (10 μm of ol/L of concentration) and XJR (10 μm of ol/L of concentration) each 0.5 μ L, Ex Taq DNA
PoLymerase (5U/ μ L) 0.5 μ L plus ddH2O to 20 μ L.Amplified reaction program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation
30s, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, 33 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.PCR primer is through 1.0%
Agarose gel electrophoresis detection after, for glue reclaim.
3.2DNA sequence and analysis
PCR primer is reclaimed with TIANgel Midi Purification Kit Ago-Gel DNA QIAquick Gel Extraction Kits, then
Transformed competence colibacillus Escherichia coli are connected with pMD 18T-vector carriers (Dalian TaKaRa companies), recombinant plasmid is handed over after purification
It is sequenced by Shanghai Sheng Gong bioengineering Co., Ltd, sequencing result analyzed with the softwares of BioEdit 7.0, and by DNA sequences
It is filed in progress Blast homologous sequence comparisons in GenBank databases.
The Molecular Identification result of 3.3 active bacterial strains
Active bacterial strain is bacillus subtilis (gene accession number through Molecular Identification：KP777596).
The ratio setting of 4 whiterot fungis, bacillus subtilis mix bacterium agent
(1) will be enlarged by the whiterot fungi after culture and expand the bacillus subtilis after culture respectively according to 1: 3,2: 3,3: 2,
3: 1 ratio carries out mixture, surveys the living bacteria count and proteinase activity and fiber of different ratio microbial inoculum after being well mixed respectively
Plain enzyme enzyme activity.
(2) 4 kinds proportioning mix bacterium agent living bacteria count and enzyme activity
The living bacteria count of 4 kinds of proportioning mix bacterium agents is shown in Table 1.As can be seen from Table 1, whiterot fungi：Bacillus subtilis (2:
3) mix bacterium agent of matched proportion density, its fungi 4.12 × 107Individual/g, bacterial number 1.18 × 109Individual/g, its effective viable bacteria
Number is 1.2212 × 109。
The cellulase and proteinase activity of 4 kinds of proportioning mix bacterium agents are shown in Table 2.As seen from Table 2, whiterot fungi: withered grass bud
The mix bacterium agent cellulase and proteinase activity of spore bacillus (2: 3) matched proportion density are respectively 77.5715U/g, 53.0000U/g,
It is above the mix bacterium agent of other matched proportion densities.
The living bacteria count testing result (individual/g) of 14 kinds of proportioning mix bacterium agents of table
The cellulase and proteinase activity (U/g) whiterot fungi of 24 kinds of proportioning mix bacterium agents of table：Withered grass
Consider with reference to the activity of living bacteria count and two kinds of enzymes, select whiterot fungi：Bacillus subtilis 2: 3 is optimum proportioning
Concentration.
The mix bacterium agent prepared using the inventive method, for hard stalk fermentation microbial inoculum, can effectively improve hard straw
The fermenting speed of stalk, so as to be agricultural production service for manufacturing organic fertilizer.The recovery profit of stalk suitable for agricultural production
With.
Brief description of the drawings
Fig. 1 is microbial inoculum of the invention and the geographical zymogenic fermentation temperature comparison diagram in day.
Embodiment
Embodiment 1：It is formulated for hard stalk fermentation microbial inoculum
Selected fungi whiterot fungi and bacteria B. subtilis are the raw material strain for being formulated for hard stalk fermentation microbial inoculum；
Fungi whiterot fungi is purchased from China General Microbiological culture presevation administrative center (China General Microbiological
Culture Collection Center, CGMCC)；Bacteria B. subtilis is voluntarily cultivated；
For the raw material strain of selection, to tobacco black shank bacterium (Phytophthora parasitica
Var.nicotiana), tobacco ralstonia solanacearum (Pseudomonas solanacearum) carries out antagonistic effect, determines it to cigarette
Careless the main pathogenic fungi antagonism；Expanded by PCR, determined dna sequence and analyze and identify the active bacterial strain molecule of selection；It is determined that
It is used for the feasibility for preparing mix bacterium agent.
Then the culture of strain is carried out：
(1) culture of fungi whiterot fungi
1. the culture of fungi whiterot fungi parent species
Iblet is soaked in advance and packed after 72h, and it is standby in being cooled down after high pressure steam sterilization 30-40min at 121 DEG C；
Then the whiterot fungi after activation is inoculated in maize culture medium, puts in constant incubator 28 DEG C of cultures, when mycelia cover with it is whole
It is standby after individual iblet；
2. the expansion culture of fungi whiterot fungi
Cultured whiterot fungi parent species are linked into wheat bran cotton seed hulls mixed culture medium, 28 DEG C of trainings in constant incubator are put
Support, after media surface covers with mycelia, throwing 1 time is turned over per 48h, until whiterot fungi is covered with whole wheat bran cotton seed hulls culture medium.Institute
It is wheat bran cotton seed hulls and mixture of the cotton seed hulls according to quality proportioning 3: 1 to state mixed culture medium, is sterilized at 121 DEG C, 30min intervals
It is obtained twice；
(2) bacteria B. subtilis culture
1. the preparation of bacillus subtilis seed liquor：By the bacillus subtilis strain of preservation in NA solid mediums, flat
16h is cultivated on plate in 37 DEG C of incubators, then picking single bacterium is fallen within seed culture medium, 37 DEG C, 170rmin-1Under the conditions of
Culture 16h is used as seed liquor.The NA solid mediums are to use beef extract 3.0g, NaCl5.0g, peptone 10.0g, agar 15-
Obtained by 20g, water 1000mL are prepared, its pH7.2-7.5；The seed culture medium is to use beef extract 3.0g, NaCl5.0g, albumen
Obtained by peptone 10.0g, water 1000mL are prepared, its pH7.2-7.5.
2. the solid fermentation of bacillus subtilis：Wheat bran and the cotton seed hulls mixture according to the composition of quality proportioning 3: 1 are existed
121 DEG C, 30min intervals sterilizing twice, obtain wheat bran cotton seed hulls mixed culture medium；Then bacillus subtilis seed liquor is pressed
10% inoculum concentration is linked into wheat bran cotton seed hulls mixed culture medium, and the addition of wheat bran cotton seed hulls mixed culture medium is 20%,
Water content is 60%, and fermentation temperature is 28 DEG C, and it is 1 time/24h to turn over throwing number of times；Realize the solid fermentation of bacillus subtilis.
Finally, whiterot fungi, the preparation of bacillus subtilis mix bacterium agent are carried out：Take and expand whiterot fungi and expansion after cultivating
Bacillus subtilis after culture is well mixed according to 2: 3 quality proportioning, then loads according to every bag 50kg amount and disappears in advance
The packaging bag of bacterium is killed, the microbial inoculum of the present invention is made.
Embodiment 2：Implement one of hard stalk fermentation microbial inoculum effect case of the present invention
In China University of Science and Technology, Hefei, Anhui Province, tobacco is tested with health research central laboratory using fermenting case
Demonstrate,prove whiterot fungi: bacillus subtilis (2: 3) is in the implementation result on hard stalk (bagasse) that becomes thoroughly decomposed.
Experiment shows：
1. whiterot fungi: bacillus subtilis (2: 3) is when for a kind of hard stalk (bagasse) organic fertilizer fermentation, fermentation
Mean temperature is respectively 33.3 DEG C, higher than the geographical zymophyte in day (pulvis) (32.4 DEG C), and is higher than 50 DEG C of temperature number of days up to 15
My god, zymophyte (pulvis) more geographical than day 2 days more.
2. whiterot fungi: the fermenting agent of bacillus subtilis (2: 3) can promote organic fertilizer in bagasse organic fertilizer production
The growth of the microorganisms such as middle fungi, bacterium, actinomyces, and the growth of the microorganisms such as Escherichia coli, mould and roundworm egg is risen
To certain inhibitory action, organic fertilizer final product quality is improved, the Escherichia coli of especially sugarcane residue organic fertilizer finished product reach
The requirement of the bio-organic fertilizer standard of NY884~2012, is better than the geographical zymophyte in day (pulvis).
Table 1 is it can be seen that whiterot fungi: bacillus subtilis (2: 3) is to organic fertilizer microorganism in fermentation process and roundworm egg number
The influence of amount：
The whiterot fungi of table 1: influence of the bacillus subtilis (2: 3) to organic fertilizer microorganism and roundworm egg quantity in fermentation process
3. add whiterot fungi: bacillus subtilis (2: 3) fermenting agent production sugarcane residue organic fertilizer finished product it is organic
The geographical zymophyte of the nutrients such as matter, total nitrogen, total phosphorus, total potassium and humic acid and day (pulvis) quite, reaches that NY525~2012 have
The requirement of machine fertilizer standard.But whiterot fungi: the fermenting agent of bacillus subtilis (2: 3) does very well in earlier fermentation.
The whiterot fungi of table 2: effect of the bacillus subtilis (2: 3) to organic fertilizer main chemical compositions in fermentation process
Embodiment 3 implements the two of the hard stalk fermentation microbial inoculum effect case of the present invention
White rot is verified using organic fertilizer scale on-site composting within 2016 at Xingyi City, Guizhou Province pig farm level ground organic fertilizer workshop
Bacterium: implementation effect of the bacillus subtilis (2: 3) in the hard stalk that becomes thoroughly decomposed (bagasse and tobacco rod mixing material) organic fertilizer composting
Really (compared with the preferable commodity microbial inoculum of using effect in current production).
The tabulation of scale demonstration reactor is bright：
(1) during hard stalk (bagasse and tobacco rod) organic fertilizer scale fermentation reactor system, whiterot fungi: bacillus subtilis
Bacterium (2: 3) can improve fermentation temperature (particularly prior fermentation temperature) and higher than 50 DEG C temperature number of days, be conducive to point of material
Solve and become thoroughly decomposed；Wherein average fermentation temperature is higher than the geographical zymophyte in day (pulvis) (45.0 DEG C), and higher than 50 DEG C temperature for 47.7 DEG C
It is 21 days, zymophyte (pulvis) more geographical than day 9 days more to spend number of days.The microbial inoculum of the present invention and the geographical zymogenic fermentation temperature pair in day
Than seeing accompanying drawing 1.
(2) whiterot fungi is utilized: the organic fertilizer quality after bacillus subtilis (2: 3) fermentation meets the NY525-2012 Ministry of Agriculture
Organic fertilizer professional standard, and total phosphorus, total potassium, total nutrient content and PH be respectively higher than the geographical zymophyte in day (pulvis) 0.22,
0.10th, 0.30,0.08 percentage point.
The whiterot fungi of table 1: influence of the bacillus subtilis (2: 3) to organic fertilizer quality after fermentation
3 whiterot fungis: bacillus subtilis (2: 3) fermentation energy improves organic fertilizer micro organism quantity after fermentation, wherein beneficial micro-
Zymophyte (pulvis) more geographical than day improves 23.2%, 97.1%, 22.2% respectively for biological bacterium, fungi, Population of Actinomycetes quantity.
The whiterot fungi of table 2: influence of the bacillus subtilis (2: 3) to organic fertilizer quantity of useful microbe after fermentation
Claims (4)
1. a kind of compound method of hard stalk fermentation microbial inoculum, comprises the following steps：
The first step, selectes strain：Using fungi whiterot fungi and bacteria B. subtilis to be formulated for hard stalk fermentation microbial inoculum
Raw material strain；The fungi whiterot fungi is purchased from China General Microbiological culture presevation administrative center (China General
Microbiological Culture Collection Center, CGMCC), fungi whiterot fungi is to cellulose degradation ability
It is stronger；The bacteria B. subtilis is voluntarily cultivated；
For the raw material strain of above-mentioned selection, it is required for by conventional means, determines it and tobacco the main pathogenic fungi antagonism is made
With the active bacterial strain molecule that identification is selected；Determine its feasibility for being used to prepare mix bacterium agent;
Second step, the culture of strain：
（1）The culture of fungi whiterot fungi
1. the culture of fungi whiterot fungi parent species
Iblet is soaked in advance and packed after 72 h, and it is standby in being cooled down after high pressure steam sterilization 30-40 min at 121 DEG C；Connect
And the whiterot fungi after activation is inoculated in maize culture medium, 28 DEG C of cultures in constant incubator are put, when mycelia is covered with entirely
It is standby after iblet；
2. the expansion culture of fungi whiterot fungi
Cultured whiterot fungi parent species are linked into wheat bran cotton seed hulls mixed culture medium, 28 DEG C of cultures in constant incubator are put,
After media surface covers with mycelia, every 48 h turns over throwing 1 time, until whiterot fungi is covered with whole wheat bran cotton seed hulls culture medium;
（2）Bacteria B. subtilis culture
1. the preparation of bacillus subtilis seed liquor：By the bacillus subtilis strain of preservation on NA solid mediums, flat board
16 h are cultivated in 37 DEG C of incubators, then picking single bacterium is fallen within seed culture medium, 37 DEG C, 170rmin-1Under the conditions of cultivate
16 h are used as seed liquor;
2. the solid fermentation of bacillus subtilis：By wheat bran with the cotton seed hulls mixture according to the composition of quality proportioning 3: 1 121
DEG C, 30 min intervals sterilizing twice, obtain wheat bran cotton seed hulls mixed culture medium；Then bacillus subtilis seed liquor is pressed 10%
Inoculum concentration be linked into wheat bran cotton seed hulls mixed culture medium, the addition of wheat bran cotton seed hulls mixed culture medium is 20%, water content
For 60%, fermentation temperature is 28 DEG C, and it is 1 time/24 h to turn over throwing number of times；Realize the solid fermentation of bacillus subtilis;
3rd step whiterot fungi, the preparation of bacillus subtilis mix bacterium agent
Take second step to expand the whiterot fungi after culture and expand the bacillus subtilis after culture to mix according to 1: 3 quality proportioning
Uniformly, then load the container of prior disinfection according to every bag 1000g amount, be stored in it is shady and cool, dry, ventilation, at lucifuge,
Avoid and poisonous substance together accumulating;
18 months shelf-lifves under the conditions of above-mentioned accumulating.
2. the method as described in claim 1, it is characterised in that determine and make to tobacco the main pathogenic fungi antagonism described in the first step
With, be to tobacco black shank bacterium, tobacco ralstonia solanacearum carry out antagonistic effect；The active bacterial strain molecule that the identification is selected includes
PCR amplifications, determined dna sequence and analysis.
3. the method as described in claim 1, it is characterised in that mixed culture medium described in second step is wheat bran cotton seed hulls and cotton
Seed shell sterilizes obtained twice according to the mixture of quality proportioning 3: 1 at 121 DEG C, 30 min intervals；The NA solid mediums
Be with beef extract 3.0g, NaCl5.0g, peptone 10.0g, agar 15-20 g, water 1000mL prepare obtained by, its pH7.2-
7.5；The seed culture medium be with the g of beef extract 3.0, NaCl5.0g, peptone 10.0g, the mL of water 1000 prepare obtained by, its
pH7.2-7.5。
4. the method as described in claim 1, it is characterised in that container can be packaging bag, pail pack, bag described in the 3rd step
One kind in tinning, vial.
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