CN106747804A - A kind of Methylotrophic gemma bacillus agent and preparation method thereof - Google Patents
A kind of Methylotrophic gemma bacillus agent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of Methylotrophic gemma bacillus agent, including mass parts raw material：10~14 parts of turf, 40~56 parts of agricultural residues powder, 13~20 parts of mineral matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter, the Methylotrophic gemma bacillus agent also Methylotrophic fermentation of bacillus liquid including 250 450mL/kg.The invention also discloses a kind of preparation method of Methylotrophic gemma bacillus agent, the Methylotrophic bacillus of present invention addition to people and animals' nonhazardous, can adjust crop microbial ecological fauna, improve crop quality.In addition, the present invention is complex carrier from turf and agricultural residues powder, turf usage amount can be both reduced, can dissolve substantial amounts of agricultural crop straw simultaneously again, the problems such as reduce the environmental pollution brought by crop straw burning.
Description
Technical field
The invention belongs to microbial manure field, specifically, be related to a kind of Methylotrophic gemma bacillus agent and its
Preparation method.
Background technology
Methylotrophic bacillus is that a class is mesophilic, aerobic, sporiferous bacillus, and the bacterium is widely present in nature
In the internal such as boundary and plant roots and stems, safety non-toxic, and the antibacterial activity with wide spectrum and extremely strong anti-adversity ability.In recent years
Come, domestic scholars to Methylotrophic bacillus in the aspect application study such as plant biological and ecological methods to prevent plant disease, pests, and erosion, growth-promoting more, Munusamy
Madhaiyan et al. (Munusamy Madhaiyan, Selvaraj Poonguzhali, Soon-WoKwon, et
al.Bacillus methylotrophicus sp.nov.,a methanol utilizing,plant-growth
promoting bacterium isolated from rice rhizosphere siol[J].IJSEM,2010,60(10):
One plant of Methylotrophic bacillus 2490-2495) is separated from The Rhizosphere of Rice soil, the bacterium can not only promote paddy rice
Growth, and the generation of paddy rice bacterial wilt can be suppressed；Zhou Dengbo et al. (Zhou Dengbo, well great waves, Tan Xin etc., 6 kinds of matrix Antagonistic Fungis
Influence [J] of the zymotic fluid to banana blight and related defensive ferment, tropical crops journal, 2013,34 (5)：947-951) from soil
The one plant of Methylotrophic Bacillus strain 4-L-16 in earth separate, there is significant prevention effect to banana blight.Lv Qian
Et al. (Lv Qian, Hu Jiangchun, Wang Nan etc., South Sea deep-sea Methylotrophic bacillus SHB114 antifungal lipopeptid activated products
Research [J], Chinese biological preventing and treating journal, 2014,30 (1)：113-120) one plant of methyl type auxotype is isolated at South Sea deep-sea
Bacillus, the bacterium is to cucumber anthracnose, Rhizoctonia solani Kuhn, cucumber fusarium axysporum etc. with stronger inhibitory action.It can be seen that,
Methylotrophic bacillus occurs in controlling disease, promotes plant growth, improve the aspect such as quality of agricultural product show it is wide
Application prospect.Also just because of this, Methylotrophic bacillus is taken a fancy to by increasing enterprise, used as production bacteria agent
Function bacterium used by.
The main component of bacteria agent is beneficial microbe living, because liquid fermentation liquid living bacteria count declines soon, no
It is easy to maintain, and logistics transportation is inconvenient, so in addition to biological foliage fertilizer, general bacteria agent is by suitable carrier adsorption
Afterwards, preparing turns into pulvis microbial inoculum or granular bacteria preparation.But there is certain requirement, it is necessary to carrier to the characteristic of carrier due to viable bacteria keep-alive
Fineness is big, with a fixed gap, and containing certain organic matter, so microbial inoculum produces big more options turf as suction for a long time
Appendix body.As the continuous expansion of bacteria agent industry, the yield of turf increase year by year, the same of certain destruction is caused to environment
When unit price also improving year by year, therefore, the suitable carrier that substitutes of selection turns into the active demand of bacteria agent production link.Stalk
Containing abundant N, P, K, organic matter and other in trace element, and aboundresources, it is cheap, be easy to get, be suitable as microbial inoculum
Carrier.In recent years, the problems such as atmosphere pollution for being brought with the increase of the stalk amount of hoarding and because of crop straw burning, frequently occurs, such as
Be applied to stalk resource in bacteria agent production by what, and this also turns into the focus of attention of bio-feritlizer industry all circles.
At present, the Methylotrophic gemma bacillus agent being related on the market or patent, its absorption carrier is more
It is turf, and turf is non-renewable resources, when long-term a large amount of exploitations have exhaustion eventually.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of Methylotrophic gemma bacillus agent and its system
On the one hand Preparation Method, the microbial inoculum adjusts crop microbial ecological fauna, improves crop quality, on the other hand reduces turf and uses
Amount, reduces the problems such as waste crop straws amount of hoarding, environmental pollution that alleviation brings by crop straw burning.
In order to solve the above-mentioned technical problem, the invention discloses a kind of Methylotrophic gemma bacillus agent, including quality
The raw material of part：10~14 parts of turf, 40~56 parts of agricultural residues powder, 13~20 parts of mineral matter, with turf, agricultural residues powder
With the quality total amount meter of mineral matter, the Methylotrophic gemma bacillus agent also Methylotrophic bud including 250-450mL/kg
Spore bacillus fermentation liquid.
Further, Methylotrophic fermentation of bacillus liquid is more than or equal to 5.5 × 10 containing living bacteria count8/ml。
Further, Methylotrophic fermentation of bacillus liquid is prepared by following steps：
1.1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded on beef peptone flat board
30 DEG C of culture 48h；Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and the liquid after 30 DEG C of culture 24h is
Required inoculation liquid；
1.2) by step 1.1) in inoculation liquid be inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium；Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min；
1.3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Enter to the 3000L fermentation tanks equipped with fermentation medium and relay supervention ferment, the same seeding tank of control condition is cultivated 28-30 hours, works as bacterium
During body sporulation 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid；
Wherein, the beef-protein medium：Beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-
7.6；
The seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/L, beef extract
2g/L；
The fermentation medium：Corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO4 0.5g/
L, CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0；
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
Further, agricultural residues powder is cassava stalk, sugarcane top, pineapple stem and leaf, maize straw, Banana Leaf, lichee
The mixture of one or more in branches and leaves, longan branches and leaves is size-reduced, and crosses the powder of straw of gained after 80 mesh sieves.
Further, mineral matter is one or more in zeolite powder, vermiculite power, diatomite, white carbon, precipitated calcium carbonate
Mixture.
The invention also discloses a kind of preparation method of Methylotrophic gemma bacillus agent, comprise the following steps：
1) Methylotrophic fermentation of bacillus liquid is prepared；
2) weigh：Following components is weighed according to mass percent：10~14 parts of turf, 40~56 parts of agricultural residues powder,
13~20 parts of mineral matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter, the Methylotrophic bacillus bacterium
The agent also Methylotrophic fermentation of bacillus liquid including 250-450mL/kg；
3) load weighted turf, Methylotrophic fermentation of bacillus liquid are poured into mixer and is adsorbed, mixing is equal
After even, toward mixer progressively adding agricultural residues powder is diluted mixing, mineral matter is eventually adding, by thing after stirring
Material is crushed, and crosses 100 mesh sieves, adjusts moisture content of material and pH value, obtains final product Methylotrophic gemma bacillus agent.
Further, Methylotrophic fermentation of bacillus liquid is prepared to be specially：
1.1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded on beef peptone flat board
30 DEG C of culture 48h；Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and the liquid after 30 DEG C of culture 24h is
Required inoculation liquid；
1.2) by step 1.1) in inoculation liquid be inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium；Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min；
1.3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Enter to the 3000L fermentation tanks equipped with fermentation medium and relay supervention ferment, the same seeding tank of control condition is cultivated 28-30 hours, works as bacterium
During body sporulation 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid.
Further, beef-protein medium：Beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-
7.6；The seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/L, beef extract 2g/L；
The fermentation medium：Corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO40.5g/L,
CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0；Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C go out
Bacterium 30 minutes.
Further, the moisture of Methylotrophic gemma bacillus agent is 6.9~7.5 less equal than 29%, pH.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention is complex carrier from turf and agricultural residues powder, and complex carrier proportion of composing has carried out strain
Experimental verification is preserved, the strain shelf-life is more than December, meets agricultural microbial agent standard GB/T 20287-2006.
Find out from the composition of product, if without 40%-56% agricultural crop straws, this partial amount is needed by turf
Supplement.This method is using agricultural crop straw and turf as carrier, it is possible to reduce 40-56% turf usage amounts, therefore, this is combined
Carrier both the holding time of extension function strain, can reduce about 50% again with assurance function microbial inoculum and the adsorbance of turf
Turf usage amount,
About 340 yuan/ton of the turf market price, and about 100-200 yuan/ton of agricultural residues powder price, use agricultural crop straw
Powder substitutes part turf, therefore can reduce product cost, therefore, the present invention has dissolved a large amount of agricultural crop straws, has both ensured product
Quality, reduces product cost, the problems such as the environmental pollution brought by crop straw burning is alleviated again.
2) present invention with the addition of Methylotrophic bacillus, and the bacterium can adjust the life of crop microorganism to people and animals' nonhazardous
State fauna, improves crop quality, and show stronger prevention effect to plant wilt disease.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The invention discloses a kind of Methylotrophic gemma bacillus agent, including mass parts raw material：10~14 parts of turf,
40~56 parts of agricultural residues powder, 13~20 parts of mineral matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter,
The Methylotrophic gemma bacillus agent also Methylotrophic fermentation of bacillus liquid including 250-450mL/kg.
Wherein, due to bacterium, living bacteria count can be gradually reduced during preservation, if Methylotrophic fermentation of bacillus
Liquid addition is small, and product living bacteria count can not reach the standard GB/T 20287-2006 of agricultural microbial agent；If addition
Amount is big, then can increase product cost.
The present invention is complex carrier from turf and agricultural residues powder, if agricultural crop straw powder content is higher than turf, no
Beneficial to the survival of bacterium, if agricultural crop straw powder content is less than turf, the purpose of agricultural crop straw of dissolving is not reached.
The addition of mineral matter, is on the one hand able to maintain that the existence of bacterium in product, on the other hand increases trace element in product
Content, addition is not enough to maintain very little the survival of bacterium, and addition is too many, can increase product cost.
The invention also discloses a kind of preparation method of Methylotrophic gemma bacillus agent, comprise the following steps：
1) Methylotrophic fermentation of bacillus liquid is prepared：
1.1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded on beef peptone flat board
30 DEG C of culture 48h；Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and the liquid after 30 DEG C of culture 24h is
Required inoculation liquid；Wherein, the beef-protein medium：Beef extract 5.0g, peptone 10.0g, agar 15-20g, pH
7.2-7.6；
1.2) by step 1.1) in inoculation liquid be inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium；Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min；The seed culture
Base：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/L, beef extract 2g/L；Wherein, initial pH value, temperature
Degree, rotating speed improve the living bacteria count amount of bacterium in zymotic fluid within the above range.
1.3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Enter to the 3000L fermentation tanks equipped with fermentation medium and relay supervention ferment, the same seeding tank of control condition is cultivated 28-30 hours, works as bacterium
During body sporulation 90%-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid；The fermentation medium：It is beautiful
Ground rice 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO40.5g/L, CaCl2.2H2O 0.1g/L,
K2HPO40.5g/L, pH 7.0；
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
2) weigh：Following components is weighed according to mass percent：10~14 parts of turf, 40~56 parts of agricultural residues powder,
13~20 parts of mineral matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter, the Methylotrophic bacillus bacterium
The agent also Methylotrophic fermentation of bacillus liquid including 250-450mL/kg；
3) load weighted turf, Methylotrophic fermentation of bacillus liquid are poured into mixer and is adsorbed, mixing is equal
After even, toward mixer progressively adding agricultural residues powder is diluted mixing, mineral matter is eventually adding, by thing after stirring
Material is crushed, and crosses 100 mesh sieves, and adjustment moisture content of material is 6.9~7.5 less equal than 29%, pH, obtains final product methylotrophy
Type gemma bacillus agent.
Embodiment 1
Formula：Methylotrophic fermentation of bacillus liquid 200L, turf 120kg, sugarcane top meal 550kg, vermiculite power
130kg。
Preparation method：Accurately weigh above-mentioned Methylotrophic fermentation of bacillus liquid, turf and pour into mixer and inhaled
It is attached, after being well mixed, then mixing is progressively diluted toward addition sugarcane top meal in mixer, vermiculite power is eventually adding, after stirring
Material is crushed, 100 mesh sieves are crossed, adjustment moisture content of material is 7.0 for 28.6%, pH, obtains final product Methylotrophic gemma
Bacillus microbial inoculum.
The Methylotrophic fermentation of bacillus liquid is prepared by the following method and obtains：
(1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded to 30 on beef peptone flat board
DEG C culture 48h.Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and inoculation liquid is obtained final product after 30 DEG C of culture 24h.Institute
Beef-protein medium is stated for beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-7.6；
(2) above-mentioned cultured inoculation liquid is inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium.Seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/
L, beef extract 2g/L；Condition of culture：Initial pH value is 7.0, and temperature is 30 DEG C, rotating speed 200r/min.
(3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Supervention ferment is relayed to the 3000L fermentation tanks equipped with fermentation medium, the same seeding tank of control condition is cultivated 28-30 hours, works as thalline
During sporulation 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid, now living bacteria count be 5.8 ×
108/ml.The fermentation medium is corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO4 0.5g/
L, CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0.
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
Embodiment 2
Formula：Methylotrophic fermentation of bacillus liquid 300L, turf 100kg, sugarcane top meal 400kg, vermiculite power
200kg。
Preparation method：Accurately weigh above-mentioned Methylotrophic fermentation of bacillus liquid, turf and pour into mixer and inhaled
It is attached, after being well mixed, then mixing is progressively diluted toward addition sugarcane top meal in mixer, vermiculite power is eventually adding, after stirring
Material is crushed, 100 mesh sieves are crossed, adjustment moisture content of material is 7.5 for 28.6%, pH, obtains final product Methylotrophic gemma
Bacillus microbial inoculum.
The Methylotrophic fermentation of bacillus liquid is prepared by the following method and obtains：
(1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded to 30 on beef peptone flat board
DEG C culture 48h.Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and inoculation liquid is obtained final product after 30 DEG C of culture 24h.Institute
Beef-protein medium is stated for beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-7.6；
(2) above-mentioned cultured inoculation liquid is inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium.Seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/
L, beef extract 2g/L；Condition of culture：Initial pH value 6.9,35 DEG C of temperature, rotating speed 180r/min.
(3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Supervention ferment is relayed to the 3000L fermentation tanks equipped with fermentation medium, the same seeding tank of control condition is cultivated 28-30 hours, works as thalline
During sporulation 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid, now living bacteria count be 5.5 ×
108/ml.The fermentation medium is corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO4 0.5g/
L, CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0.
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
Embodiment 3
Formula：Methylotrophic fermentation of bacillus liquid 300L, turf 140kg, sugarcane top meal 560kg, vermiculite power
150kg。
Preparation method：Accurately weigh above-mentioned Methylotrophic fermentation of bacillus liquid, turf and pour into mixer and inhaled
It is attached, after being well mixed, then mixing is progressively diluted toward addition sugarcane top meal in mixer, vermiculite power is eventually adding, after stirring
Material is crushed, 100 mesh sieves are crossed, adjustment moisture content of material is 7.3 for 29.2%, pH, obtains final product Methylotrophic gemma
Bacillus microbial inoculum.
The Methylotrophic fermentation of bacillus liquid is prepared by the following method and obtains：
(1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded to 30 on beef peptone flat board
DEG C culture 48h.Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, and inoculation liquid is obtained final product after 30 DEG C of culture 24h.Institute
Beef-protein medium is stated for beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-7.6；
(2) above-mentioned cultured inoculation liquid is inoculated in the 300L seeding tanks equipped with seed culture medium, connecing bacterium amount is
10%, fermented after connecing bacterium.Seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/
L, beef extract 2g/L；Condition of culture：Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min.
(3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, now adds the zymotic fluid in seeding tank
Supervention ferment is relayed to the 3000L fermentation tanks equipped with fermentation medium, the same seeding tank of control condition is cultivated 28-30 hours, works as thalline
During sporulation 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid, now living bacteria count be 6.6 ×
108/ml.The fermentation medium is corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO4 0.5g/
L, CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0.
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
Embodiment 4
Formula：Methylotrophic fermentation of bacillus liquid 450L, turf 150kg, sugarcane top meal 625kg, vermiculite power
225kg。
Preparation method：Accurately weigh above-mentioned Methylotrophic fermentation of bacillus liquid, turf and pour into mixer and inhaled
It is attached, after being well mixed, then mixing is progressively diluted toward addition sugarcane top meal in mixer, vermiculite power is eventually adding, after stirring
Material is crushed, 100 mesh sieves are crossed, adjustment moisture content of material is 6.9 for 29%, pH, obtains final product Methylotrophic gemma bar
Bacteria agent.
The Methylotrophic fermentation of bacillus liquid is prepared by the method for embodiment one, and living bacteria count is 5.5
×108/ml。
Technique effect of the invention is illustrated with reference to specific experimental program：
Choose 3 consistent leaf Muskmelon Seedlings of growing way to be transplanted in basin alms bowl, after transplanting 7 days, muskmelon seedling is carried out to connect bacterium treatment.
Treatment 1：Pathogen of Fusarium Wilt is first inoculated with, the microbial inoculum prepared in applying embodiment 1,2,3 respectively again every 1 day, labeled as embodiment
1st, embodiment 2, embodiment 3；Treatment 2：The microbial inoculum prepared in embodiment 1,2,3 is first applied respectively, and droop disease was inoculated every 1 day
Opportunistic pathogen, labeled as embodiment I, embodiment II, embodiment III；Control treatment：Pathogen of Fusarium Wilt is only inoculated with, labeled as CK.Often
10 basins of individual treatment, per 1 plant of muskmelon seedling of basin.Pathogen inoculum concentration is 107Cfu/mL spore suspension 10ml, Methylotrophic bud
Spore bacillus microbial inoculum amount of application bacterium is 1g/ basins.Connect bacterium 7 days afterwards, record muskmelon incidence, calculate its disease index and prevent relatively
Effect is controlled, statistics is shown in Table 1.
Muskmelon Fusarium wilt incidence is divided into 5 grades by standard：0 grade：Complete stool is disease-free；1 grade：Plant blade face table below 1/4
Now wilt symptom；2 grades：The blade face of plant 1/4 to 1/2 shows wilting symptom；3 grades：The blade face of plant more than 1/2 shows wilting symptom：4
Level：Complete stool is wilted dead.
Disease index=[Σ (the sick level strain number × disease series)/(representative of the treatment Potted orchard summation × morbidity most heavy duty
Numerical value)] × 100.
With respect to prevention effect=[(control disease index-treatment disease index)/control disease index] × 100%.
Prevention effect of the Methylotrophic gemma bacillus agent of the present invention of table 1 to Muskmelon Fusarium wilt
Result above shows, first applies Methylotrophic gemma bacillus agent and be followed by the muskmelon disease index that pathogen is processed
The treatment for first connecing and microbial inoculum being applied after pathogen is less than, illustrates that Methylotrophic gemma bacillus agent is to muskmelon to a certain extent
Droop has certain prevention effect.In addition, applying before either first pathogen or afterwards microbial inoculum, it is right to be above
According to prevention effect.It can be seen that, Methylotrophic gemma bacillus agent of the invention has certain prevention and preventing and treating to Muskmelon Fusarium wilt
Effect.
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
Claims (9)
1. a kind of Methylotrophic gemma bacillus agent, it is characterised in that the raw material including mass parts：10~14 parts of turf, agriculture
40~56 parts of crop material powder, 13~20 parts of mineral matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter, should
The Methylotrophic gemma bacillus agent also Methylotrophic fermentation of bacillus liquid including 250-450mL/kg.
2. Methylotrophic gemma bacillus agent according to claim 1, it is characterised in that described Methylotrophic bud
Spore bacillus fermentation liquid is more than or equal to 5.5 × 10 containing living bacteria count8/ml。
3. Methylotrophic gemma bacillus agent according to claim 1, it is characterised in that the Methylotrophic gemma
Bacillus fermentation liquid is prepared by following steps：
1.1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded to 30 DEG C on beef peptone flat board
Culture 48h；Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, needed for the liquid after 30 DEG C of culture 24h is
Inoculation liquid；
1.2) by step 1.1) in inoculation liquid be inoculated in the 300L seeding tanks equipped with seed culture medium, connect bacterium amount for 10%,
Fermented after connecing bacterium；Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min；
1.3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, is now added to the zymotic fluid in seeding tank
3000L fermentation tanks relaying supervention ferment equipped with fermentation medium, the same seeding tank of control condition is cultivated 28-30 hours, when thalline bud
When spore forms 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid；
Wherein, the beef-protein medium：Beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-7.6；
The seed culture medium：Glucose 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/L, beef extract 2g/L；
The fermentation medium：Corn flour 30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO40.5g/L,
CaCl2.2H2O 0.1g/L, K2HPO40.5g/L, pH 7.0；
Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
4. Methylotrophic gemma bacillus agent according to claim 1, it is characterised in that described agricultural residues powder
It is one or more in cassava stalk, sugarcane top, pineapple stem and leaf, maize straw, Banana Leaf, lichee branches and leaves, longan branches and leaves
Mixture is size-reduced, and crosses the powder of straw of gained after 80 mesh sieves.
5. Methylotrophic gemma bacillus agent according to claim 1, it is characterised in that the mineral matter is zeolite
The mixture of one or more in powder, vermiculite power, diatomite, white carbon, precipitated calcium carbonate.
6. a kind of preparation method of Methylotrophic gemma bacillus agent, it is characterised in that comprise the following steps：
1) Methylotrophic fermentation of bacillus liquid is prepared；
2) weigh：Following components is weighed according to mass percent：10~14 parts of turf, 40~56 parts of agricultural residues powder, mineral
13~20 parts of matter, in terms of the quality total amount of turf, agricultural residues powder and mineral matter, the Methylotrophic gemma bacillus agent is also
Methylotrophic fermentation of bacillus liquid including 250-450mL/kg；
3) load weighted turf, Methylotrophic fermentation of bacillus liquid are poured into mixer and is adsorbed, after being well mixed,
Agricultural residues powder is progressively added toward mixer and is diluted mixing, be eventually adding mineral matter, enter material after stirring
Row is crushed, and crosses 100 mesh sieves, adjusts moisture content of material and pH value, obtains final product Methylotrophic gemma bacillus agent.
7. the preparation method of Methylotrophic gemma bacillus agent according to claim 6, it is characterised in that described system
Standby Methylotrophic fermentation of bacillus liquid is specially：
1.1) the Methylotrophic Bacillus strain that will be stored under -4 DEG C of environment is seeded to 30 DEG C on beef peptone flat board
Culture 48h；Picking single bacterium colony is seeded in beef extract-peptone fluid nutrient medium, needed for the liquid after 30 DEG C of culture 24h is
Inoculation liquid；
1.2) by step 1.1) in inoculation liquid be inoculated in the 300L seeding tanks equipped with seed culture medium, connect bacterium amount for 10%,
Fermented after connecing bacterium；Initial pH value 6.9-7.1,28-35 DEG C of temperature, rotating speed 180-220r/min；
1.3) when seeding tank ferments 9-10 hours, thalli growth is in logarithmic phase, is now added to the zymotic fluid in seeding tank
3000L fermentation tanks relaying supervention ferment equipped with fermentation medium, the same seeding tank of control condition is cultivated 28-30 hours, when thalline bud
When spore forms 90-95%, terminate fermentation, obtain final product Methylotrophic fermentation of bacillus liquid.
8. the preparation method of Methylotrophic gemma bacillus agent according to claim 7, it is characterised in that the beef
Cream protein culture medium：Beef extract 5.0g, peptone 10.0g, agar 15-20g, pH 7.2-7.6；The seed culture medium：Portugal
Grape sugar 10g/L, peptone 2g/L, MgSO40.5g/L, yeast extract 2g/L, beef extract 2g/L；The fermentation medium：Corn flour
30.0g/L, beancake powder 20.0g/L, MgSO40.2g/L, NaH2PO40.5g/L, CaCl2.2H2O 0.1g/L, K2HPO4
0.5g/L, pH 7.0；Each medium sterilization condition is：0.1-0.15MPa, 121 DEG C sterilize 30 minutes.
9. the preparation method of Methylotrophic gemma bacillus agent according to claim 6, it is characterised in that the methyl
The moisture of auxotype gemma bacillus agent is 6.9~7.5 less equal than 29%, pH.
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