CN107711891A - A kind of method prevented and treated matrimony vine root rot and extremely set - Google Patents

A kind of method prevented and treated matrimony vine root rot and extremely set Download PDF

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CN107711891A
CN107711891A CN201710947311.2A CN201710947311A CN107711891A CN 107711891 A CN107711891 A CN 107711891A CN 201710947311 A CN201710947311 A CN 201710947311A CN 107711891 A CN107711891 A CN 107711891A
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matrimony vine
bacillus
bacillus subtilis
decomposed
root rot
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CN107711891B (en
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赵钢勇
孙海远
顾欣燕
刘亚利
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Tianjin Development Zone Kunhe Biotechnology Co ltd
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The present invention relates to a kind of method prevented and treated matrimony vine root rot and extremely set, using complex micro organism fungicide and decomposed protein biology organic fertilizer collective effect preventing and treating matrimony vine root rot, application method is that obtained biological organic fertilizer base manure is after the matrimony vine after field planting applies decomposed mushroom residue；Then the matrimony vine after being colonized applies complex micro organism fungicide in 25 centimetres of plant radius every year；The complex micro organism fungicide by bacillus amyloliquefaciens MES812 tunning and bacillus subtilis MES810 tunnings by volume 1:13 mix.The complex micro organism fungicide of the present invention and being used cooperatively for decomposed protein biology organic fertilizer, both the growth of pathogen can have been suppressed, the soil organism and mineral nutrient content can be increased again, it is effectively improved soil texture, nutrient situation and micro-ecological environment, prevention matrimony vine root rot is extremely set, increase matrimony vine survival rate, improve Lycium barbarum. L Quality, increase yield of medlar.

Description

A kind of method prevented and treated matrimony vine root rot and extremely set
Technical field
The invention belongs to field of agricultural biotechnology, is related to a kind of method prevented and treated matrimony vine root rot and extremely set.
Background technology
Matrimony vine is the perennial machaka of Solanaceae, is important medicine-food two-purpose economic plant resources.In recent years, with matrimony vine Planting scale constantly expands, and the generation of matrimony vine multiple diseases also aggravates year by year, and wherein root rot is one of Major Diseases.Boxthorn root Maize ear rot is a kind of disease by soil infection, and pathogen is four kinds of Fusariumsps.Fusarium oxysporum F.oxysporum, eggplant disease sickle Spore bacterium F.solani, homochromy Fusariumsp F.concolor and Fusarium moniliforme F.moniliforme, what wherein pathogenicity was most strong is Fusarium oxysporum, it is secondly Fusaium solani, homochromy Fusariumsp and beading sickle bacterium are weak pathogenic bacteria.After morbidity, diseased plant root and Root neck occurs to rot, and cane vascular bundle becomes brown.Due to root-rot, blade diminishes, and lacks gloss, and branch is downgraded, fruit slight of stature； Or blade flavescence is wilted, plant is gradually withered when serious.Due to the infringement of root rot, the matrimony vine death rate is every year in 2%-6%, most For the important place incidence of disease up to 40%, the death rate is up to 25%, plants and produces to matrimony vine and brings very big loss.Preventing and treating matrimony vine root-rot at present Die of illness tree method mainly spray insecticide, rational application of fertilizers, the mode of increasing application phosphorus potassium fertilizer, waste time and energy, pollute environment, it is poisonous to have Do harm to and effective controlling disease can not occur from root, therefore green biological prevention is increasingly paid close attention to by people, Do not find the report in terms of preventing and treating matrimony vine root rot with fertilizer using microbial bacterial agent also at present.
The content of the invention
The purpose of the present invention is the deficiency for matrimony vine root rot of the prior art preventing and treating, there is provided one kind preventing and treating boxthorn root Complex micro organism fungicide that maize ear rot is extremely set and its preparation method and application.The present invention utilizes the micro- life of main effect in complex micro organism fungicide Large area breeding of the thing in soil, the growth of antagonism pathogen, effectively suppresses or kills harmful microorganism in soil, be beneficial The breeding and development of microorganism are provided with force environment, and boxthorn root is effectively prevented and treated by the continuous formation of beneficial microbe metabolite The generation of maize ear rot, prevention matrimony vine are extremely set, and increase matrimony vine survival rate.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of complex micro organism fungicide prevented and treated matrimony vine root rot and extremely set, the complex micro organism fungicide is by solution starch gemma Bacillus (Bacillus amyloliquefaciens) MES812 tunning and bacillus subtilis (Bacillus Subtilis) MES810 tunnings by volume 1:1-3 is mixed；
Bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is 2017 8 The moon 10, deposit number CGMCCNo.14515.
The B. subtilis cell is shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.
Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.
Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.
Bacillus amyloliquefaciens MES812 is that my company research staff divides in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County From, by plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut Piece, crystallized purple dyeing, microscopy, is defined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, use Bacillus amyloliquefaciens carry out flat board face-off experiment to it, 30 DEG C of cultures, find that the bacillus can significantly inhibit after 4 days Growth of pathogenic bacteria, there is wider antibacterial band, be named as MES812, identified by identification mechanism of the Ministry of Agriculture, MES812 is Bacillus amyloliquefaciens.
Bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Preferably, withered grass gemma in the tunning of bacillus subtilis (Bacillus subtilis) MES810 Bacillus (Bacillus subtilis) MES810 viable count is no less than 2.0*1010cFu/g；
Starch is solved in described solution starch bud bacillus (Bacillus amyloliquefaciens) MES812 tunning Bacillus (Bacillus amyloliquefaciens) MES812 viable count is no less than 2.0*1010cFu/g。
Preferably, the complex micro organism fungicide that the preventing and treating matrimony vine root rot is extremely set is liquid bacterial agent.
As preferable technical scheme：
The preparation method for the complex micro organism fungicide extremely set present invention also offers above-mentioned preventing and treating matrimony vine root rot, specifically Comprise the following steps：
1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
(1) actication of culture：In 250mL conical flasks load 50mL sterilized waters, picking bacillus subtilis slant strains in In conical flask, shake up, take 1-2mL liquid into 500mL Kolle flasks, load the training of 100mL beef extract-peptones in Kolle flask in advance Base is supported, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, must be activated Strain；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and it is sterile to pour into 50-100mL Bacteria suspension is made in water, scraping bacterium tire, and its whole is poured into the seed bottle equipped with the sterilized water that 100-300mL and temperature are 4 DEG C, Obtain primary seed solution；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL Primary seed solution is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24h；Obtain secondary seed nutrient solution；
(4) ferment tank：Load fermenter volume 50%-60% bacillus subtilis hair in 3000L fermentation tanks Ferment culture medium, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solutions are all inoculated in withered grass bud and embraced In bacillus viable bacteria fermentation culture medium, control tank pressure 0.02MPa-0.07MPa, after 30 DEG C of -38 DEG C of culture 24-36h, discharging obtains The tunning of bacillus subtilis；
2nd, the preparation of bacillus amyloliquefaciens tunning：
(1) actication of culture：Load 50mL sterilized waters, picking bacillus amyloliquefaciens slant strains in 250mL conical flasks In conical flask, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 100mL beef extract-peptones in Kolle flask in advance Culture medium, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, obtains work Change strain；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and it is sterile to pour into 50-100mL Bacteria suspension is made in water, scraping bacterium tire, and its whole is poured into the seed bottle equipped with the sterilized water that 100-300mL and temperature are 4 DEG C, Obtain primary seed solution；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL Primary seed solution is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24h；Obtain secondary seed nutrient solution；
(4) ferment tank：Load fermenter volume 50%-60% bacillus subtilis hair in 3000L fermentation tanks Ferment culture medium, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solutions are all inoculated in solution starch bud In spore bacillus viable bacteria fermentation culture medium, control tank pressure 0.02MPa-0.07MPa, after 30 DEG C of -38 DEG C of culture 24-36h, discharge To bacillus amyloliquefaciens tunning；
3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:1-3 is mixed Close, obtain the complex micro organism fungicide that the preventing and treating matrimony vine root rot is extremely set.
Preferably, beef-protein medium phase used by the bacillus amyloliquefaciens and bacillus subtilis Together, specifically, as mass fraction：Beef extract 0.5%-3%, peptone 0.5%-3%, sodium chloride 0.2-1%, nutrient agar 1.5%-2.0%, surplus are water, medium pH 6.0-7.5.
Preferably, used by the bacillus amyloliquefaciens and bacillus subtilis secondary seed medium into split-phase Together, specifically, as mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, defoamer 0.2 ‰ -2 ‰, surplus is water, and medium pH is adjusted to 6.0-7.5, It is and standby in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.
Preferably, the composition of fermentation medium is identical used by the bacillus amyloliquefaciens and bacillus subtilis, Specifically, as mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, defoamer 0.2 ‰ -2 ‰, surplus is water, and the pH of culture medium is adjusted to 6.0- 7.5, and in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.
The complex micro organism fungicide that the preventing and treating matrimony vine root rot of the present invention is extremely set can be used alone, and can also coordinate decomposed It is more preferable that protein biology organic fertilizer is used together effect, is specially：
The preparation method of decomposed protein biology organic fertilizer：
(1) raw material：Mushroom residue
(2) feed：Mushroom residue (both decomposed bacterium of two kinds of decomposed strains of Candida and bacillus subtilis will be inoculated with It is purchased from Baoding and is just accord with bio tech ltd) and fermentation tunnel is inserted by the mushroom residue being inoculated with is well mixed, needed before charging Clear up ventilating system tuyere and cleaning airduct buildup, it is ensured that ventilation is smooth.
(3) fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, Air intake heat source is closed, is divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, ensures that ventilation can every time Stop ventilation within 20 minutes after blowing through decomposed material.It can be gradually risen after the of short duration slightly decline of decomposed material temperature, when temperature reaches 40 degree, Start constant ventilation, when temperature, which reaches more than 60, to be spent, continuing decomposed 5 days temperatures above can be remarkably decreased.
(4) discharge：When temperature of charge is reduced to 30 degree (summers decline close to outdoor natural temperature), and do not have in decomposed material Stop decomposed, normal discharging when having stink and ammonia odor, and having light yeast-leavened paste flavor taste.
(5) starch gemma bar is conciliate after discharging by the tunning for sieving, crushing, adding bacillus subtilis MES810 Bacterium MES812 tunning, stirring, packaging, decomposed protein biology organic fertilizer finished product is obtained, wherein, bacillus subtilis MES810 Addition in discharge product of tunning, bacillus amyloliquefaciens MES812 tunning be 0.5-1wt%.
The method that the preventing and treating matrimony vine root rot of the present invention is extremely set, this method are for the fertilising after matrimony vine field planting and planting tube Reason, all Cultivate administration modes before field planting are carried out in a conventional manner：
(1) base manure is applied：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure.The corruption of above-mentioned preparation is applied every year The white biological organic fertilizer of soft-boiled eggs is as base manure, and fertilising radius is 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth.
(2) complex micro organism fungicide is applied：Matrimony vine after field planting applies above-mentioned preparation in 25 centimetres of plant radius every year Complex micro organism fungicide, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once.
(3) trim：Trimming in First Year July is less than with trunk angle 30 degree strong twice, during trimming pair after matrimony vine field planting Branch, wipe out in time.Continue to trim after the branch that clip is formed.
(4) pour water：7th, want diversion flood irrigation 2-3 times within 8 two months, pour water not cross furrow face, filling is defined thoroughly.If soil water-retaining is poor, Fill once, draining is poor more, few to fill once.
(5) top dressing：Late July, weeding, top dressing.Top dressing is based on nitrogenous fertilizer, every mu 10-15 kilograms.It is decomposed due to application of Protein biology organic fertilizer and complex micro organism fungicide, during top dressing, dose reduces 20%-40% than traditional topdressing amount.
(6) pluck：Into August, the after ripening of fruit elder generation, manually plucked.
Beneficial effect：
The method method that the preventing and treating matrimony vine root rot of the present invention is extremely set is simple to operate, prevents and treats matrimony vine root rot positive effect, Matrimony vine root rot extremely tree rate is significantly reduced, at the same time it can also improve the yield and quality of matrimony vine.
Main effect microorganism is in soil in the complex micro organism fungicide that the method for present invention preventing and treating matrimony vine root rot utilizes Large area is bred, the growth of antagonism pathogen, is effectively suppressed or is killed harmful microorganism in soil, is bred for beneficial microorganism Force environment is provided with development, the generation of matrimony vine root rot is effectively prevented and treated by the continuous formation of beneficial microbe metabolite, Prevention matrimony vine is extremely set, and increases matrimony vine survival rate.
The decomposed protein biology organic fertilizer that the present invention applies can promote soil granular while the soil organism is increased Structure is formed, improved soil, and combines distinctive farming method, and the inventive method is in preventing and treating matrimony vine soil-borne disease, reduction While matrimony vine is extremely set, soil microenvironment has been repaired, has improved soil fertility, has added yield of medlar and quality.
By the microbial inoculum of the present invention complex micro organism fungicide for preventing matrimony vine root rot, prevention effect is notable, and without public affairs It is harmful, pollution-free, inexpensive, be easy to large area to use.Fluid present invention microbial inoculum is easy to drip irrigation, micro- spray, punching to apply, easy to use.
Embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, art technology Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Fixed scope.
Embodiment 1
A kind of complex micro organism fungicide prevented and treated matrimony vine root rot and extremely set, the complex micro organism fungicide is by solution starch gemma Bacillus (Bacillus amyloliquefaciens) MES812 tunning and bacillus subtilis (Bacillus Subtilis) MES810 tunnings by volume 1:3 mix；
Bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is 2017 8 The moon 10, deposit number CGMCCNo.14515.
The B. subtilis cell is shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.
Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.
Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.
Bacillus amyloliquefaciens MES812 is that my company research staff divides in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County From, by plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut Piece, crystallized purple dyeing, microscopy, is defined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, use Bacillus amyloliquefaciens carry out flat board face-off experiment to it, 30 DEG C of cultures, find that the bacillus can significantly inhibit after 4 days Growth of pathogenic bacteria, there is wider antibacterial band, be named as MES812, identified by identification mechanism of the Ministry of Agriculture, MES812 is Bacillus amyloliquefaciens.
Bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The preparation method for the complex micro organism fungicide extremely set present invention also offers above-mentioned preventing and treating matrimony vine root rot, specifically Comprise the following steps：
1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 120 DEG C are damp and hot Sterilized 30min, and Kolle flask is placed under the conditions of 34 DEG C and cultivates 30h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 80mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 200mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 200mL one-levels Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 55% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 0.05MPa, 35 DEG C of culture 30h；Obtain bacillus subtilis (Ehrenberg) Cohn fermented product.In fermentation a period of time, such as after 24 hours, every 40 minutes from three In the bottle of angle sampling carry out microscopy, the gemma in the visual field and total thalline number are counted, and calculate gemma rate (gemma rate (%)= Grown spore number/(grown spore number+thalline number) × 100)；Gemma rate stops fermented and cultured when reaching 90%, discharging obtains withered Careless bacillus liquid preparation.
Wherein, beef-protein medium is specifically, as mass fraction：Beef extract 3%, peptone 0.5%, chlorination Sodium 1%, nutrient agar 1.5%, surplus are water, medium pH 7.0.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 3%, disodium hydrogen phosphate 0.25 ‰, sodium dihydrogen phosphate 2 ‰, defoamer 1 ‰, surplus is water, and medium pH is adjusted to 7.0, and 118 DEG C moist heat sterilization 30min is standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 1%, corn flour 4%, bean cake powder 3%, phosphorus Sour disodium hydrogen 0.3 ‰, sodium dihydrogen phosphate 4 ‰, defoamer 1.5 ‰, surplus are water, and the pH of culture medium is adjusted to 7.1, and at 118 DEG C Moist heat sterilization 35min.
2nd, the preparation of bacillus amyloliquefaciens tunning：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C are damp and hot Sterilized 35min, and Kolle flask is placed under the conditions of 32 DEG C and cultivates 36h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 50mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100 one-level kinds Sub- liquid is inoculated in secondary seed medium；24h is cultivated at 30 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 50% in 3000L fermentation tanks Base, 150L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium, control tank pressure 0.02MPa, 36h is cultivated at 30 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 0.5%, peptone 3%, chlorine Change sodium 0.2%, nutrient agar 1.5%%, surplus is water, and the pH of culture medium is 6.0, and standby in 116 DEG C of moist heat sterilization 20min With.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 5%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 0.5 ‰, defoamer 0.2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C of moist heat sterilization 40min are standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 5%, Disodium hydrogen phosphate 0.1 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and at 115 DEG C Moist heat sterilization 40min.
3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:3 mixing, Obtain the composite fluid microbial inoculum that the preventing and treating matrimony vine root rot is extremely set.
The complex micro organism fungicide that the preventing and treating matrimony vine root rot of the present invention is extremely set can be used alone, and can also coordinate decomposed It is more preferable that protein biology organic fertilizer is used together effect, is specially：
The preparation method of decomposed protein biology organic fertilizer：
(1) raw material：Mushroom residue
(2) feed：Mushroom residue (both decomposed bacterium of two kinds of decomposed strains of Candida and bacillus subtilis will be inoculated with It is purchased from Baoding and is just accord with bio tech ltd) and fermentation tunnel is inserted by the mushroom residue being inoculated with is well mixed, needed before charging Clear up ventilating system tuyere and cleaning airduct buildup, it is ensured that ventilation is smooth.
(3) fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, Air intake heat source is closed, is divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, ensures that ventilation can every time Stop ventilation within 20 minutes after blowing through decomposed material.It can be gradually risen after the of short duration slightly decline of decomposed material temperature, when temperature reaches 40 degree, Start constant ventilation, when temperature, which reaches more than 60, to be spent, continuing decomposed 5 days temperatures above can be remarkably decreased.
(4) discharge：When temperature of charge is reduced to 30 degree (summers decline close to outdoor natural temperature), and do not have in decomposed material Stop decomposed, normal discharging when having stink and ammonia odor, and having light yeast-leavened paste flavor taste.
(5) starch gemma bar is conciliate after discharging by the tunning for sieving, crushing, adding bacillus subtilis MES810 Bacterium MES812 tunning, stirring, packaging, decomposed protein biology organic fertilizer finished product is obtained, wherein, bacillus subtilis MES810 Addition in discharge product of tunning, bacillus amyloliquefaciens MES812 tunning be 0.6wt%.
The method that the preventing and treating matrimony vine root rot of the present invention is extremely set, this method are for the fertilising after matrimony vine field planting and planting tube Reason, all Cultivate administration modes before field planting are carried out in a conventional manner：
(1) base manure is applied：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure.The corruption of above-mentioned preparation is applied every year The white biological organic fertilizer of soft-boiled eggs is as base manure, and fertilising radius is 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth.
(2) complex micro organism fungicide is applied：Matrimony vine after field planting applies above-mentioned preparation in 25 centimetres of plant radius every year Complex micro organism fungicide, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once.
(3) trim：Trimming in First Year July is less than with trunk angle 30 degree strong twice, during trimming pair after matrimony vine field planting Branch, wipe out in time.Continue to trim after the branch that clip is formed.
(4) pour water：7th, want diversion flood irrigation 2-3 times within 8 two months, pour water not cross furrow face, filling is defined thoroughly.If soil water-retaining is poor, Fill once, draining is poor more, few to fill once.
(5) top dressing：Late July, weeding, top dressing.Top dressing is based on nitrogenous fertilizer, every mu 10-15 kilograms.It is decomposed due to application of Protein biology organic fertilizer and complex micro organism fungicide, during top dressing, dose reduces 20%-40% than traditional topdressing amount.
(6) pluck：Into August, the after ripening of fruit elder generation, manually plucked.
Embodiment 2
A kind of complex micro organism fungicide prevented and treated matrimony vine root rot and extremely set, the complex micro organism fungicide is by solution starch gemma Bacillus (Bacillus amyloliquefaciens) MES812 tunning and bacillus subtilis (Bacillus Subtilis) MES810 tunnings by volume 1:1 mixes；
Bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is 2017 8 The moon 10, deposit number CGMCCNo.14515.
The B. subtilis cell is shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.
Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.
Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.
Bacillus amyloliquefaciens MES812 is that my company research staff divides in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County From, by plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut Piece, crystallized purple dyeing, microscopy, is defined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, use Bacillus amyloliquefaciens carry out flat board face-off experiment to it, 30 DEG C of cultures, find that the bacillus can significantly inhibit after 4 days Growth of pathogenic bacteria, there is wider antibacterial band, be named as MES812, identified by identification mechanism of the Ministry of Agriculture, MES812 is Bacillus amyloliquefaciens.
Bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The preparation method for the complex micro organism fungicide extremely set present invention also offers above-mentioned preventing and treating matrimony vine root rot, specifically Comprise the following steps：
1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C are damp and hot Sterilized 35min, and Kolle flask is placed under the conditions of 32 DEG C and cultivates 36h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 50mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100 one-level kinds Sub- liquid is inoculated in secondary seed medium；24h is cultivated at 30 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 50% in 3000L fermentation tanks Base, 150L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 0.02MPa, 30 DEG C of culture 36h；Discharging obtains bacillus subtilis liquid fermentation production.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 0.5%, peptone 3%, chlorine Change sodium 0.2%, nutrient agar 1.5%%, surplus is water, and the pH of culture medium is 6.0, and standby in 116 DEG C of moist heat sterilization 20min With.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 5%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 0.5 ‰, defoamer 0.2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C of moist heat sterilization 40min are standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 5%, Disodium hydrogen phosphate 0.1 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and at 115 DEG C Moist heat sterilization 40min.
2nd, the preparation of bacillus amyloliquefaciens tunning：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 120 DEG C are damp and hot Sterilized 30min, and Kolle flask is placed under the conditions of 34 DEG C and cultivates 30h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 80mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 200mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 200mL one-levels Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 55% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium, control tank pressure 0.05MPa, 30h is cultivated at 35 DEG C；Obtain zymotic fluid, i.e. bacillus amyloliquefaciens tunning.In fermentation a period of time, such as after 24 hours, Every 40 minutes sampling progress microscopies from triangular flask, the gemma in the visual field and total thalline number are counted, and calculate gemma Rate (gemma rate (%)=grown spore number/(grown spore number+thalline number) × 100)；Gemma rate stops fermentation when reaching 90% Culture, discharging obtain bacillus amyloliquefaciens tunning.
Wherein, beef-protein medium is specifically, as mass fraction：Beef extract 3%, peptone 0.5%, chlorination Sodium 1%, nutrient agar 1.5%, surplus are water, medium pH 7.0.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 3%, disodium hydrogen phosphate 0.25 ‰, sodium dihydrogen phosphate 2 ‰, defoamer 1 ‰, surplus is water, and medium pH is adjusted to 7.0, and 118 DEG C moist heat sterilization 30min is standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 1%, corn flour 4%, bean cake powder 3%, phosphorus Sour disodium hydrogen 0.3 ‰, sodium dihydrogen phosphate 4 ‰, defoamer 1.5 ‰, surplus are water, and the pH of culture medium is adjusted to 7.1, and at 118 DEG C Moist heat sterilization 35min.
3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:1 mixing, Obtain the complex micro organism fungicide that the preventing and treating matrimony vine root rot is extremely set.
The complex micro organism fungicide that the preventing and treating matrimony vine root rot of the present invention is extremely set can be used alone, and can also coordinate decomposed It is more preferable that protein biology organic fertilizer is used together effect, is specially：
The preparation method of decomposed protein biology organic fertilizer：
(1) raw material：Mushroom residue
(2) feed：Mushroom residue (both decomposed bacterium of two kinds of decomposed strains of Candida and bacillus subtilis will be inoculated with It is purchased from Baoding and is just accord with bio tech ltd) and fermentation tunnel is inserted by the mushroom residue being inoculated with is well mixed, needed before charging Clear up ventilating system tuyere and cleaning airduct buildup, it is ensured that ventilation is smooth.
(3) fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, Air intake heat source is closed, is divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, ensures that ventilation can every time Stop ventilation within 20 minutes after blowing through decomposed material.It can be gradually risen after the of short duration slightly decline of decomposed material temperature, when temperature reaches 40 degree, Start constant ventilation, when temperature, which reaches more than 60, to be spent, continuing decomposed 5 days temperatures above can be remarkably decreased.
(4) discharge：When temperature of charge is reduced to 30 degree (summers decline close to outdoor natural temperature), and do not have in decomposed material Stop decomposed, normal discharging when having stink and ammonia odor, and having light yeast-leavened paste flavor taste.
(5) starch gemma bar is conciliate after discharging by the tunning for sieving, crushing, adding bacillus subtilis MES810 Bacterium MES812 tunning, stirring, packaging, decomposed protein biology organic fertilizer finished product is obtained, wherein, bacillus subtilis MES810 Addition in discharge product of tunning, bacillus amyloliquefaciens MES812 tunning be 0.8wt%.
The method that the preventing and treating matrimony vine root rot of the present invention is extremely set, this method are for the fertilising after matrimony vine field planting and planting tube Reason, all Cultivate administration modes before field planting are carried out in a conventional manner：
(1) base manure is applied：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure.The corruption of above-mentioned preparation is applied every year The white biological organic fertilizer of soft-boiled eggs is as base manure, and fertilising radius is 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth.
(2) complex micro organism fungicide is applied：Matrimony vine after field planting applies above-mentioned preparation in 25 centimetres of plant radius every year Complex micro organism fungicide, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once.
(3) trim：Trimming in First Year July is less than with trunk angle 30 degree strong twice, during trimming pair after matrimony vine field planting Branch, wipe out in time.Continue to trim after the branch that clip is formed.
(4) pour water：7th, want diversion flood irrigation 2-3 times within 8 two months, pour water not cross furrow face, filling is defined thoroughly.If soil water-retaining is poor, Fill once, draining is poor more, few to fill once.
(5) top dressing：Late July, weeding, top dressing.Top dressing is based on nitrogenous fertilizer, every mu 10-15 kilograms.It is decomposed due to application of Protein biology organic fertilizer and complex micro organism fungicide, during top dressing, dose reduces 20%-40% than traditional topdressing amount.
(6) pluck：Into August, the after ripening of fruit elder generation, manually plucked.
Embodiment 3
A kind of complex micro organism fungicide prevented and treated matrimony vine root rot and extremely set, the complex micro organism fungicide is by solution starch gemma Bacillus (Bacillus amyloliquefaciens) MES812 tunning and bacillus subtilis (Bacillus Subtilis) MES810 tunnings by volume 1:2 mix；
Bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is 2017 8 The moon 10, deposit number CGMCCNo.14515.
The B. subtilis cell is shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.
Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.
Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.
Bacillus amyloliquefaciens MES812 is that my company research staff divides in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County From, by plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut Piece, crystallized purple dyeing, microscopy, is defined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, use Bacillus amyloliquefaciens carry out flat board face-off experiment to it, 30 DEG C of cultures, find that the bacillus can significantly inhibit after 4 days Growth of pathogenic bacteria, there is wider antibacterial band, be named as MES812, identified by identification mechanism of the Ministry of Agriculture, MES812 is Bacillus amyloliquefaciens.
Bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The preparation method for the complex micro organism fungicide extremely set present invention also offers above-mentioned preventing and treating matrimony vine root rot, specifically Comprise the following steps：
1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 2mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 121 DEG C are damp and hot Sterilized 20min, and Kolle flask is placed under the conditions of 36 DEG C and cultivates 24h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 300mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 300mL one-levels Seed liquor is inoculated in secondary seed medium；12h is cultivated at 38 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 60% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, control tank pressure 0.07MPa, 38 DEG C of culture 24h；Discharging obtains bacillus subtilis (Ehrenberg) Cohn fermented product.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 3%, peptone 0.5%, chlorine Change sodium 1%, nutrient agar 2.0%, surplus is water, and the pH of culture medium is 7.5, and standby in 121 DEG C of moist heat sterilization 35min.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 1%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C moist heat sterilization 40min is standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 1%, phosphorus Sour disodium hydrogen 0.5 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and wet at 122 DEG C Heat sterilization 20min.
2nd, the preparation of bacillus amyloliquefaciens tunning：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1.5mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 117 DEG C are wet Heat sterilization 25min, Kolle flask is placed under the conditions of 33 DEG C and cultivates 28h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 60mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 150mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 250mL one-levels Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 58% in 3000L fermentation tanks Base, 116 DEG C of moist heat sterilization 30min, 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium In, control tank pressure 0.05MPa, 28h is cultivated at 34 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 1.5%, peptone 0.25%, sodium chloride 0.8%, nutrient agar 1.8%, surplus is water, and the pH of culture medium is 6.8, and in 120 DEG C of moist heat sterilizations 24min is standby.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 2.5%, disodium hydrogen phosphate 0.4 ‰, sodium dihydrogen phosphate 3.4 ‰, defoamer 1.5 ‰, surplus is water, and medium pH is adjusted to 7.2, and It is standby in 118 DEG C of moist heat sterilization 35min.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 1.4%, corn flour 2.7%, bean cake powder 2.5%, disodium hydrogen phosphate 0.2 ‰, sodium dihydrogen phosphate 2.4 ‰, defoamer 1.4 ‰, surplus is water, and the pH of culture medium is adjusted to 6.9, And in 118 DEG C of moist heat sterilization 35min.
3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:2 mixing, Obtain the complex micro organism fungicide that the preventing and treating matrimony vine root rot is extremely set.
2.0* is no less than by the content of bacillus subtilis in bacillus subtilis microbial inoculum product made from the above method 1010cFu/g。
The complex micro organism fungicide that the preventing and treating matrimony vine root rot of the present invention is extremely set can be used alone, and can also coordinate decomposed It is more preferable that protein biology organic fertilizer is used together effect, is specially：
The preparation method of decomposed protein biology organic fertilizer：
(1) raw material：Mushroom residue
(2) feed：Mushroom residue (both decomposed bacterium of two kinds of decomposed strains of Candida and bacillus subtilis will be inoculated with It is purchased from Baoding and is just accord with bio tech ltd) and fermentation tunnel is inserted by the mushroom residue being inoculated with is well mixed, needed before charging Clear up ventilating system tuyere and cleaning airduct buildup, it is ensured that ventilation is smooth.
(3) fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, Air intake heat source is closed, is divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, ensures that ventilation can every time Stop ventilation within 20 minutes after blowing through decomposed material.It can be gradually risen after the of short duration slightly decline of decomposed material temperature, when temperature reaches 40 degree, Start constant ventilation, when temperature, which reaches more than 60, to be spent, continuing decomposed 5 days temperatures above can be remarkably decreased.
(4) discharge：When temperature of charge is reduced to 30 degree (summers decline close to outdoor natural temperature), and do not have in decomposed material Stop decomposed, normal discharging when having stink and ammonia odor, and having light yeast-leavened paste flavor taste.
(5) starch gemma bar is conciliate after discharging by the tunning for sieving, crushing, adding bacillus subtilis MES810 Bacterium MES812 tunning, stirring, packaging, decomposed protein biology organic fertilizer finished product is obtained, wherein, bacillus subtilis MES810 Addition in discharge product of tunning, bacillus amyloliquefaciens MES812 tunning be 0.5wt%.
The method that the preventing and treating matrimony vine root rot of the present invention is extremely set, this method are for the fertilising after matrimony vine field planting and planting tube Reason, all Cultivate administration modes before field planting are carried out in a conventional manner：
(1) base manure is applied：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure.The corruption of above-mentioned preparation is applied every year The white biological organic fertilizer of soft-boiled eggs is as base manure, and fertilising radius is 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth.
(2) complex micro organism fungicide is applied：Matrimony vine after field planting applies above-mentioned preparation in 25 centimetres of plant radius every year Complex micro organism fungicide, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once.
(3) trim：Trimming in First Year July is less than with trunk angle 30 degree strong twice, during trimming pair after matrimony vine field planting Branch, wipe out in time.Continue to trim after the branch that clip is formed.
(4) pour water：7th, want diversion flood irrigation 2-3 times within 8 two months, pour water not cross furrow face, filling is defined thoroughly.If soil water-retaining is poor, Fill once, draining is poor more, few to fill once.
(5) top dressing：Late July, weeding, top dressing.Top dressing is based on nitrogenous fertilizer, every mu 10-15 kilograms.It is decomposed due to application of Protein biology organic fertilizer and complex micro organism fungicide, during top dressing, dose reduces 20%-40% than traditional topdressing amount.
(6) pluck：Into August, the after ripening of fruit elder generation, manually plucked.
Embodiment 4
A kind of complex micro organism fungicide prevented and treated matrimony vine root rot and extremely set, the complex micro organism fungicide is by solution starch gemma Bacillus (Bacillus amyloliquefaciens) MES812 tunning and bacillus subtilis (Bacillus Subtilis) MES810 tunnings by volume 1:1 mixes；
Bacillus subtilis (Bacillus subtilis) MES810, it is deposited in China General Microbiological strain guarantor Administrative center is hidden, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is August in 2017 10, protects It is CGMCCNo.14514 to hide numbering；
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MES812, it is deposited in Chinese common micro- Biological inoculum preservation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is 2017 8 The moon 10, deposit number CGMCCNo.14515.
The B. subtilis cell is shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-3.5 μm, gemma Middle life, ellipse, sporangium are not expanded.On broth bouillon, 48h bacterium colonies are circular, and edge is irregular, dry tack free, flat, White.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithinase；Propionate utilizes, breast Sugared fermentation and acid.
Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplified production, stripe size and withered grass bud Spore bacillus (Bacillus subtilis) is identical.
Bacillus subtilis MES810 is that my company research staff separates in the potato field of Zhangjiakou, is passed through Plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut into slices, crystallized purple Dyeing, microscopy, is defined as bacillus subtilis.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, with bacillus to it Carry out flat board face-off experiment, 30 DEG C culture, find that the bacillus can significantly inhibit growth of pathogenic bacteria after 4 days, occur compared with Wide antibacterial band, is named as MES810, identifies that MES810 is bacillus subtilis by identification mechanism of the Ministry of Agriculture.
Bacillus amyloliquefaciens MES812 is that my company research staff divides in the matrimony vine ground of peaceful Xia Zhongning city Zhongning County From, by plate streaking, cultivated 24 hours at 32 DEG C, bevel strain, 4 DEG C of refrigerations.Then picking single bacterium colony, cut Piece, crystallized purple dyeing, microscopy, is defined as bacillus amyloliquefaciens.Using the matrimony vine pine root fungus sickle-like bacteria of separation as target, use Bacillus amyloliquefaciens carry out flat board face-off experiment to it, 30 DEG C of cultures, find that the bacillus can significantly inhibit after 4 days Growth of pathogenic bacteria, there is wider antibacterial band, be named as MES812, identified by identification mechanism of the Ministry of Agriculture, MES812 is Bacillus amyloliquefaciens.
Bacillus amyloliquefaciens morphological feature：Bacillus amyloliquefaciens are shaft-like, Gram-positive, and size is 0.6-0.8 μm of * 2.0-4.5 μm, raw in gemma, ellipse, sporangium is not expanded.On broth bouillon, 48h bacterium colonies are circular, Micro- protuberance, edge is irregular, and color is dark.Positive reaction：Catalase；Oxidizing ferment；Hydrolysis starch.Negative reaction：Anaerobic growth, lecithin Lipase；Propionate utilizes, lactose fermentation production acid.Multiplexed PCR amplification is carried out using special primer, the bacterial strain produces unique amplification Product, stripe size are identical with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The preparation method for the complex micro organism fungicide extremely set present invention also offers above-mentioned preventing and treating matrimony vine root rot, specifically Comprise the following steps：
1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 1.5mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 117 DEG C are wet Heat sterilization 25min, Kolle flask is placed under the conditions of 33 DEG C and cultivates 28h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 60mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 150mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 250mL one-levels Seed liquor is inoculated in secondary seed medium；20h is cultivated at 35 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 58% in 3000L fermentation tanks Base, 116 DEG C of moist heat sterilization 30min, 200L secondary seed solutions are all inoculated in bacillus subtilis viable bacteria fermentation culture medium, Tank pressure 0.05MPa is controlled, 28h is cultivated at 34 DEG C；Discharging obtains bacillus subtilis liquid preparation.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 1.5%, peptone 0.25%, sodium chloride 0.8%, nutrient agar 1.8%, surplus is water, and the pH of culture medium is 6.8, and in 120 DEG C of moist heat sterilizations 24min is standby.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 1.5%, corn flour 3%, bean cake powder 2.5%, disodium hydrogen phosphate 0.4 ‰, sodium dihydrogen phosphate 3.4 ‰, defoamer 1.5 ‰, surplus is water, and medium pH is adjusted to 7.2, and It is standby in 118 DEG C of moist heat sterilization 35min.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 1.4%, corn flour 2.7%, bean cake powder 2.5%, disodium hydrogen phosphate 0.2 ‰, sodium dihydrogen phosphate 2.4 ‰, defoamer 1.4 ‰, surplus is water, and the pH of culture medium is adjusted to 6.9, And in 118 DEG C of moist heat sterilization 35min.
2nd, the preparation of bacillus amyloliquefaciens tunning：
(1) actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking slant strains are shaken in conical flask It is even, 2mL liquid is taken into 500mL Kolle flasks, loads 100mL beef-protein mediums in Kolle flask in advance, 121 DEG C are damp and hot Sterilized 20min, and Kolle flask is placed under the conditions of 36 DEG C and cultivates 24h, obtains activated spawn；
(2) preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 300mL and temperature for 4 DEG C of sterilized water, obtains one-level kind Sub- liquid；
(3) preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 300mL one-levels Seed liquor is inoculated in secondary seed medium；12h is cultivated at 38 DEG C；Obtain secondary seed nutrient solution；
(4) ferment tank：Load the fermentation of bacillus subtilis culture of fermenter volume 60% in 3000L fermentation tanks Base, 200L secondary seed solutions are all inoculated in bacillus amyloliquefaciens viable bacteria fermentation culture medium, control tank pressure 0.07MPa, 24h is cultivated at 38 DEG C；Discharging obtains bacillus amyloliquefaciens tunning.
Wherein, the composition of beef-protein medium is, as mass fraction：Beef extract 3%, peptone 0.5%, chlorine Change sodium 1%, nutrient agar 2.0%, surplus is water, and the pH of culture medium is 7.5, and standby in 121 DEG C of moist heat sterilization 35min.
Wherein, the composition of secondary seed medium is, as mass fraction：Glucose 0.3%, corn flour 5%, bean cake powder 1%, disodium hydrogen phosphate 0.5 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus is water, and medium pH is adjusted to 7.5, and 122 DEG C moist heat sterilization 40min is standby.
Wherein, the composition of fermentation medium is, as mass fraction：Glucose 2%, corn flour 1%, bean cake powder 1%, phosphorus Sour disodium hydrogen 0.5 ‰, sodium dihydrogen phosphate 5 ‰, defoamer 2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0, and wet at 122 DEG C Heat sterilization 20min.
3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:1 mixing, Obtain the complex micro organism fungicide that the preventing and treating matrimony vine root rot is extremely set.
2.0* is no less than by the content of bacillus subtilis in bacillus subtilis microbial inoculum product made from the above method 1010cFu/g。
The complex micro organism fungicide that the preventing and treating matrimony vine root rot of the present invention is extremely set can be used alone, and can also coordinate decomposed It is more preferable that protein biology organic fertilizer is used together effect, is specially：
The preparation method of decomposed protein biology organic fertilizer：
(1) raw material：Mushroom residue
(2) feed：Mushroom residue (both decomposed bacterium of two kinds of decomposed strains of Candida and bacillus subtilis will be inoculated with It is purchased from Baoding and is just accord with bio tech ltd) and fermentation tunnel is inserted by the mushroom residue being inoculated with is well mixed, needed before charging Clear up ventilating system tuyere and cleaning airduct buildup, it is ensured that ventilation is smooth.
(3) fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, Air intake heat source is closed, is divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, ensures that ventilation can every time Stop ventilation within 20 minutes after blowing through decomposed material.It can be gradually risen after the of short duration slightly decline of decomposed material temperature, when temperature reaches 40 degree, Start constant ventilation, when temperature, which reaches more than 60, to be spent, continuing decomposed 5 days temperatures above can be remarkably decreased.
(4) discharge：When temperature of charge is reduced to 30 degree (summers decline close to outdoor natural temperature), and do not have in decomposed material Stop decomposed, normal discharging when having stink and ammonia odor, and having light yeast-leavened paste flavor taste.
(5) starch gemma bar is conciliate after discharging by the tunning for sieving, crushing, adding bacillus subtilis MES810 Bacterium MES812 tunning, stirring, packaging, obtain decomposed protein biology organic fertilizer finished product.Wherein, bacillus subtilis MES810 Addition in discharge product of tunning, bacillus amyloliquefaciens MES812 tunning be 0.6wt%.
The method that the preventing and treating matrimony vine root rot of the present invention is extremely set, this method are for the fertilising after matrimony vine field planting and planting tube Reason, all Cultivate administration modes before field planting are carried out in a conventional manner：
(1) base manure is applied：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure.The corruption of above-mentioned preparation is applied every year The white biological organic fertilizer of soft-boiled eggs is as base manure, and fertilising radius is 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth.
(2) complex micro organism fungicide is applied：Matrimony vine after field planting applies above-mentioned preparation in 25 centimetres of plant radius every year Complex micro organism fungicide, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once.
(3) trim：Trimming in First Year July is less than with trunk angle 30 degree strong twice, during trimming pair after matrimony vine field planting Branch, wipe out in time.Continue to trim after the branch that clip is formed.
(4) pour water：7th, want diversion flood irrigation 2-3 times within 8 two months, pour water not cross furrow face, filling is defined thoroughly.If soil water-retaining is poor, Fill once, draining is poor more, few to fill once.
(5) top dressing：Late July, weeding, top dressing.Top dressing is based on nitrogenous fertilizer, every mu 10-15 kilograms.It is decomposed due to application of Protein biology organic fertilizer and complex micro organism fungicide, during top dressing, dose reduces 20%-40% than traditional topdressing amount.
(6) pluck：Into August, the after ripening of fruit elder generation, manually plucked.
2nd, Function detection
1st, the antagonism test to bacillus subtilis MES810 produced by the present invention to matrimony vine root rot
For examination matrimony vine root rot germ source：The withered diseased plant in peaceful Xia Zhongning city Zhongning County is picked up from matrimony vine root rot germ strain, Isolated and purified through company researcher, be accredited as Fusarium oxysporum (F.oxysporum), Pathogenic Tests show as strong cause a disease Power.
(1) bacillus subtilis MES810 flat board face-off experiment：
Matrimony vine root rot germ is coated in beef extract-peptone flat board center first, then by the withered grass bud after obtained activation Spore bacillus MES810 points are connected on away from the centimeters of indicator bacteria bacterium piece 2.0, if blank control.33 degrees Celsius incubated, treats blank pair During according to whole culture dish will be covered with, the control increment (colony radius) and processing increment of matrimony vine root rot germ are measured (the suppression growth radius after inoculation MES810), with antagonism bacteriostasis rate (bacteriostasis rate (%)=(control increment-processing life Long amount)/control increment * 100) fungistatic effect is characterized, shown in table 1 specific as follows.
Antagonistic effect results of the bacillus subtilis MES810 of table 1 to matrimony vine root rot
Strain name Compare increment (mm) Handle increment (mm) Bacteriostasis rate (%)
MES810 28.0 5.0 82.1
Experimental result：Show that bacillus subtilis MES810 reaches to the inhibiting rate of matrimony vine root rot from the result of table 1 82.1%；10.0 millimeters of transparent antibacterial bandwidth.Illustrate that bacillus subtilis MES810 has obvious suppression to matrimony vine root rot Make and use, there is the Biocontrol Potential of preventing and treating matrimony vine root rot.
(2) bacillus amyloliquefaciens MES812 flat board face-off experiment：
Matrimony vine root rot germ is coated in beef extract-peptone flat board center first, then by the solution starch after obtained activation Bacillus MES812 points are connected on away from the centimeters of indicator bacteria bacterium piece 2.0, if blank control.33 degrees Celsius incubated, treats blank When control will cover with whole culture dish, the control increment (colony radius) and processing increment of matrimony vine root rot germ are measured (the suppression growth radius after inoculation MES812), with antagonism bacteriostasis rate (bacteriostasis rate (%)=(control increment-processing life Long amount)/control increment * 100) fungistatic effect is characterized, shown in table 2 specific as follows.
Antagonistic effect results of the bacillus amyloliquefaciens MES812 of table 2 to matrimony vine root rot
Strain name Compare increment (mm) Handle increment (mm) Bacteriostasis rate (%)
MES812 28.0 4.0 85.7
Experimental result：Show that bacillus amyloliquefaciens MES812 reaches to the inhibiting rate of matrimony vine root rot from the result of table 2 85.7%；9.0 millimeters of transparent antibacterial bandwidth.Illustrate that bacillus amyloliquefaciens MES812 has obvious suppression to matrimony vine root rot Make and use, there is the Biocontrol Potential of preventing and treating matrimony vine root rot.
3rd, the complex micro organism fungicide to the present invention and decomposed protein biology organic fertilizer are to the Field information of matrimony vine root rot Effect test
In peaceful Xia Zhongning city Zhongning County, selection matrimony vine root rot extremely sets serious plot, designs 4 processing, and each processing is set 3 repetitions are put, each matrimony vine tree for repeating to select 20 growing ways essentially identical.
Processing 1：Around every matrimony vine tree plant in 25 centimeters radius at the beginning of 3 months, using decomposed protein biology organic fertilizer, 10 Kilogram/, 30 centimetres of fertilization depth.March and September apply complex micro organism fungicide, and (viable count is no less than 2.0*1010CFu/g), 50ml/ every time, applied after 300 times of dilution in 25 centimetres of plant radius.Other way to manages are the same as 3.4.Wherein, decomposed albumen Biological organic fertilizer and complex micro organism fungicide are according to made from the preparation method of embodiment 4.
Processing 2：March and September apply complex micro organism fungicide, and (viable count is no less than 2.0*1010CFu/g), each 50ml/ , applied after 300 times of dilution in 25 centimetres of plant radius.Other way to manages are the same as 3.4.
Processing 3：Around every matrimony vine tree plant in 25 centimeters radius at the beginning of 3 months, using decomposed protein biology organic fertilizer, 10 Kilogram/, 30 centimetres of fertilization depth.Other way to manages are the same as 3.4.
Processing 4：Decomposed protein biology organic fertilizer and complex micro organism fungicide are not applied, and other way to manages are the same as processing 1.
(2) experimental result is shown in Table 3
The contrast test of table 3 preventing and treating matrimony vine root rot extremely tree and effect of increasing production
(3) experimental result：As can be seen that the decomposed protein biology provided using the inventive method is had from the result of table 3 After machine fertilizer and complex micro organism fungicide, the matrimony vine droop incidence of disease reduces 63.34 percentage points, and yield adds 293.3 Percentage point.Preventing disease production-increasing effect using decomposed protein biology organic fertilizer and complex micro organism fungicide is best, using only compound micro- Bacteria agent effect is taken second place, but higher than the effect using only decomposed protein biology organic fertilizer.
Complex micro organism fungicide contains bacillus subtilis and the solution starch gemma bar of antagonism matrimony vine Pathogens Causing Root Rot Disease Bacterium, after being manured into soil, the growth and breeding of two kinds of probiotics can suppress the growth of pathogen, reduce the activity of pathogen, so as to Dissemination of the pathogen to plant is reduced, effectively prevents the generation of matrimony vine root rot.Bacillus subtilis conciliates starch simultaneously The growth of bacillus can increase soil beneficial microorganism species and activity, improve soil microenvironment, so as to promote Chinese holly Absorption of the Qi root system to soil nutrient elements, promote matrimony vine plant healthy growth, increasing both production and income.
The use of decomposed protein biology organic fertilizer can increase soil organic matter content, improve soil granular structure, promote Beneficial microorganism is bred in soil, soil is maintained good microbiota, improved soil micro-ecological environment, is promoted soil The release of mineral nutrient, and then promote the healthy growth of matrimony vine, improve Lycium barbarum. L Quality, increase yield of medlar.
Being used cooperatively for complex micro organism fungicide and decomposed protein biology organic fertilizer, can both suppress the growth of pathogen, The soil organism and mineral nutrient content can be increased again, be effectively improved soil texture, nutrient situation and micro-ecological environment, prevented Matrimony vine root rot is extremely set, and increases matrimony vine survival rate, improves Lycium barbarum. L Quality, increases yield of medlar.
Microbial bacterial agent of the present invention has nontoxic, noresidue, does not suppress the characteristics of growth and dosage are few,.Meanwhile The inventive method can promote absorption of the plant to nutrient, reduce fertilizer and pesticide usage amount, effectively reduction crop agriculture is residual, protection ring Reach preventing and treating soil-borne disease while border, increase the effect of matter volume increase.
The foregoing is only a preferred embodiment of the present invention, but it is not limited to embodiment of above.The guarantor of the present invention Shield scope is not limited thereto, any to be familiar with those skilled in the art in the technical scope of present disclosure, according to this hair Bright technical scheme and its inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.

Claims (7)

  1. A kind of 1. method prevented and treated matrimony vine root rot and extremely set, it is characterised in that given birth to using complex micro organism fungicide and decomposed albumen Organic fertilizer collective effect preventing and treating matrimony vine root rot is extremely set, and specific application method is：
    （1）Using base manure：Matrimony vine after field planting is poured water, after weeding, starts to apply base manure；The base manure is that decomposed protein biology has Machine fertilizer, fertilising radius be 25 centimetres, 5-10 kilograms/, 30-40 centimetres of fertilization depth；The decomposed protein biology organic fertilizer is Obtained biological organic fertilizer after decomposed mushroom residue；
    （2）Using complex micro organism fungicide：Matrimony vine after field planting applies composite microbial bacteria in 25 centimetres of plant radius every year Agent, every is applied 100ml, is poured after 300 times of dilution, and March, October are respectively once；Then carry out field management to matrimony vine into It is ripe；
    The complex micro organism fungicide is by bacillus amyloliquefaciens（Bacillus amyloliquefaciens）MES812 hair Ferment product and bacillus subtilis (Bacillus subtilis) MES810 tunning by volume 1:1-3 mix and Into；
    Bacillus subtilis (Bacillus subtilis) MES810, is deposited in China General Microbiological culture presevation pipe Reason center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is August in 2017 10, and preservation is compiled Number it is CGMCCNo.14514；
    The bacillus amyloliquefaciens（Bacillus amyloliquefaciens）MES812, it is deposited in China General Microbiological Culture presevation administrative center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and preservation date is August 10 in 2017 Day, deposit number CGMCCNo.14515.
  2. A kind of 2. method prevented and treated matrimony vine root rot and extremely set according to claim 1, it is characterised in that the decomposed albumen The preparation process of biological organic fertilizer is：
    （1）Raw material：Mushroom residue is used as raw material；
    （2）Charging：The mushroom residue for being inoculated with two kinds of decomposed strains of Candida and bacillus subtilis is inserted into fermentation tunnel；
    （3）Fermentation maturity：Closure of a tunnel gate, air blower and air intake heat source are opened, after decomposed material reaches 28 DEG C, closed Air intake heat source, divulged information using intermittent aeration mode, at interval of 2 hours ventilation 1-2 hours, stop ventilation；When temperature reaches At 40 degree, start constant ventilation, when temperature, which reaches more than 60, to be spent, after continuing decomposed more than 5 days, temperature is begun to decline；
    （4）Discharging：When temperature drops to 30 degree, and it is destitute of smell in decomposed material and ammonia odor, and has light yeast hair Stop decomposed, normal discharging during the paste flavor taste of ferment；
    （5）By sieving, crushing, add bacillus subtilis MES810 tunning and bacillus amyloliquefaciens after discharging MES812 tunning, stirring, packaging, obtains decomposed protein biology organic fertilizer finished product.
  3. 3. a kind of method prevented and treated matrimony vine root rot and extremely set according to claim 1, it is characterized in that：The bacillus subtilis Bacillus subtilis (Bacillus subtilis) in bacterium (Bacillus subtilis) MES810 tunning MES810 viable count is no less than 2.0*1010cFu/g；
    The solution starch bud bacillus（Bacillus amyloliquefaciens）Starch gemma is solved in MES812 tunning Bacillus（Bacillus amyloliquefaciens）MES812 viable count is no less than 2.0*1010cFu/g；
    The complex micro organism fungicide is liquid bacterial agent.
  4. A kind of 4. method prevented and treated matrimony vine root rot and extremely set according to claim 1, it is characterised in that the composite microbial Thing microbial inoculum preparation process, specifically includes following steps：
    1st, the preparation of bacillus subtilis (Ehrenberg) Cohn fermented product：
    （1）Actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking bacillus subtilis slant strains are in taper In bottle, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 100mL beef-protein mediums in Kolle flask in advance, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, obtains activated spawn；
    （2）The preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 50-100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100-300mL and temperature for 4 DEG C of sterilized water, obtains one Level seed liquor；
    （3）The preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL one-levels Seed liquor is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24 h；Obtain secondary seed nutrient solution；
    （4）Ferment tank：Load fermenter volume 50%-60% fermentation of bacillus subtilis culture in 3000L fermentation tanks Base, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, 150L or 200L secondary seed solutions are all inoculated in bacillus subtilis and lived In bacteria fermentation culture medium, control tank presses 0.02 MPa-0.07 MPa, and after 30 DEG C of -38 DEG C of culture 24-36h, discharging obtains withered grass The tunning of bacillus；
    2nd, the preparation of bacillus amyloliquefaciens tunning：
    （1）Actication of culture：Load 50mL sterilized waters in 250mL conical flasks, picking bacillus amyloliquefaciens slant strains are in cone In shape bottle, shake up, take 1-2mL liquid into 500mL Kolle flasks, load 100mL beef extract-peptone cultures in Kolle flask in advance Base, 116 DEG C of -121 DEG C of moist heat sterilization 20-35min, Kolle flask is placed under the conditions of 32 DEG C -36 DEG C and cultivates 24-36h, must activate bacterium Kind；
    （2）The preparation of primary seed solution：The Kolle flask that activated spawn is filled by more than takes out, and pours into 50-100mL sterilized waters, scrapes Take bacterium tire that bacteria suspension is made, its whole is poured into equipped with the seed bottle of 100-300mL and temperature for 4 DEG C of sterilized water, obtains one Level seed liquor；
    （3）The preparation of secondary seed solution：150L secondary seed mediums are housed in 300L fermentation tanks, by 100-300mL one-levels Seed liquor is inoculated in secondary seed medium；In 30 DEG C of -38 DEG C of culture 12-24 h；Obtain secondary seed nutrient solution；
    （4）Ferment tank：Load fermenter volume 50%-60% fermentation of bacillus subtilis culture in 3000L fermentation tanks Base, 115 DEG C of -122 DEG C of moist heat sterilization 20-40min, bacillus amyloliquefaciens are all inoculated in by 150L or 200L secondary seed solutions In viable bacteria fermentation culture medium, control tank presses 0.02 MPa-0.07 MPa, and after 30 DEG C of -38 DEG C of culture 24-36h, discharging is solved Bacillus amyloliquefacienses tunning；
    3rd, by the tunning of bacillus amyloliquefaciens and the tunning of bacillus subtilis by volume 1:1-3 is mixed, and is obtained The complex micro organism fungicide extremely set to the preventing and treating matrimony vine root rot.
  5. A kind of 5. method prevented and treated matrimony vine root rot and extremely set according to claim 4, it is characterised in that the solution starch bud Beef-protein medium is identical used by spore bacillus and bacillus subtilis, specifically, as mass fraction：Beef extract 0.5%-3%, peptone 0.5%-3%, sodium chloride 0.2-1%, nutrient agar 1.5%-2.0%, surplus are water, medium pH 6.0- 7.5。
  6. A kind of 6. method prevented and treated matrimony vine root rot and extremely set according to claim 4, it is characterised in that the solution starch bud The composition of secondary seed medium is identical used by spore bacillus and bacillus subtilis, specifically, as mass fraction：Grape Sugared 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, Defoamer 0.2 ‰ -2 ‰, surplus are water, and medium pH is adjusted to 6.0-7.5, and in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min It is standby.
  7. A kind of 7. method prevented and treated matrimony vine root rot and extremely set according to claim 4, it is characterised in that the solution starch bud The composition of fermentation medium is identical used by spore bacillus and bacillus subtilis, specifically, as mass fraction：Glucose 0.3%-2%, corn flour 1%-5%, bean cake powder 1%-5%, disodium hydrogen phosphate 0.1 ‰ -0.5 ‰, sodium dihydrogen phosphate 0.5 ‰ -5 ‰, disappear Infusion 0.2 ‰ -2 ‰, surplus are water, and the pH of culture medium is adjusted to 6.0-7.5, and in 115 DEG C of -122 DEG C of moist heat sterilization 20-40min.
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CN109362786A (en) * 2018-11-16 2019-02-22 山西果信农业科技发展有限公司 A kind of large cherry root rot control agent and control method
CN109593681A (en) * 2018-12-28 2019-04-09 天津坤禾生物科技集团股份有限公司 A kind of composite bacteria agent and its preparation method and application for preventing and treating aquaculture cyanobacterial bloom
CN109673671A (en) * 2019-01-22 2019-04-26 河北省农林科学院植物保护研究所 A kind of microorganism seed inorganic agent and preparation method for preventing and treating potato verticillium wilt
CN111892460A (en) * 2020-07-24 2020-11-06 天津开发区坤禾生物技术有限公司 Bio-organic fertilizer for improving soil quality of saline-alkali soil and application thereof
CN114853543A (en) * 2022-04-20 2022-08-05 青海省湟中县海宁合资化肥厂 Liquid fertilizer for preventing and treating root rot of Chinese wolfberry, preparation method, prevention and treatment method and application

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CN105255777A (en) * 2015-11-12 2016-01-20 湖南农业大学 Method for producing bio-organic fertilizer through pig manure and mushroom residues
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CN108383604A (en) * 2018-04-11 2018-08-10 河南沃特威生物科技有限公司 A kind of biological organic fertilizer and preparation method thereof of prevention fruit tree root rot
CN109362786A (en) * 2018-11-16 2019-02-22 山西果信农业科技发展有限公司 A kind of large cherry root rot control agent and control method
CN109593681A (en) * 2018-12-28 2019-04-09 天津坤禾生物科技集团股份有限公司 A kind of composite bacteria agent and its preparation method and application for preventing and treating aquaculture cyanobacterial bloom
CN109673671A (en) * 2019-01-22 2019-04-26 河北省农林科学院植物保护研究所 A kind of microorganism seed inorganic agent and preparation method for preventing and treating potato verticillium wilt
CN111892460A (en) * 2020-07-24 2020-11-06 天津开发区坤禾生物技术有限公司 Bio-organic fertilizer for improving soil quality of saline-alkali soil and application thereof
CN114853543A (en) * 2022-04-20 2022-08-05 青海省湟中县海宁合资化肥厂 Liquid fertilizer for preventing and treating root rot of Chinese wolfberry, preparation method, prevention and treatment method and application

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