CN105695342B - Koning trichoderma bacterium TG-72 and its application in Aspergillus flavus biological control - Google Patents

Koning trichoderma bacterium TG-72 and its application in Aspergillus flavus biological control Download PDF

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CN105695342B
CN105695342B CN201610149130.0A CN201610149130A CN105695342B CN 105695342 B CN105695342 B CN 105695342B CN 201610149130 A CN201610149130 A CN 201610149130A CN 105695342 B CN105695342 B CN 105695342B
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koning trichoderma
aspergillus flavus
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李培武
姚彦坡
丁小霞
张奇
张文
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The present invention relates to koning trichoderma bacterium TG-72 and its applications in Aspergillus flavus biological control.Koning trichoderma bacterium TG-72 was preserved in China typical culture collection center (CCTCC) on January 7th, 2016, and deposit number is CCTCC NO:M 2016015.Koning trichoderma bacterium TG-72 is to separate to obtain from the peanut pod of the south china town Ba Bu, finds that the bacterial strain bacteriostasis is stronger through indoor face-off antagonistic experiment, the antibacterial removing toxic substances experiment of living body and field controling test, antibacterial detoxifying effect is good and stablizes, it is easy culture, it is pollution-free, it is environmentally safe.After the bacterial manure is manured into soil, Antagonistic Fungi can quickly be bred in crop rhizosphere and long term survival, forms dominant microflora, can effectively prevent harm of the aspergillus flavus to peanut.

Description

Koning trichoderma bacterium TG-72 and its application in Aspergillus flavus biological control
Technical field
The present invention relates to beneficial to fungi application field, and in particular to koning trichoderma bacterium TG-72 and its in Aspergillus flavus biology Application in prevention and treatment.
Background technique
The endotoxin contamination as caused by aspergillus fungi (especially aspergillus flavus) is referred to as Aspergillus endotoxin contamination, host range Extensively.Aspergillus especially aspergillus flavus brings serious harm to the safety in production of peanut, corn, rice and Soybean and Other Crops, leads every year The economic loss of cause is up to tens billion of dollars.Aspergillus fungi especially Aspergillus flavus can generate the extremely strong mycotoxin of toxicity. Aflatoxin is strong carcinogenic, the hypertoxicity mycotoxin of one kind of extensive pollution agricultural product, including B, G and M race, and wherein B1 is most general All over and toxicity it is most strong, be 10 times of potassium cyanide, 68 times of arsenic, carcinogenicity is 10000 times of Isotox, human or animal's food It is even dead with will lead to body disease after the food or feed by endotoxin contamination, it seriously threatens consumer health and life is pacified Entirely, agricultural industry development and international trade are restricted.Therefore, reinforce in the agricultural product such as China's peanut, corn and rice aspergillus flavus and The prevention and control of endotoxin contamination are very urgent.
Currently, chemical prevention occupies consequence in the prevention and treatment to Aspergillus flavus.Chemical prevention is not only at high cost, And environment easy to pollute, pathogen also easily generates drug resistance even drug resistance to chemical agent while prevention and control pathogen.Therefore, Reinforce the prevention and control of Aspergillus flavus biology, be increasingly becoming focus concerned by people and hot spot, this develops to China's agricultural industry and agricultural production The raising of quality has great importance.
The country is using in bio-control factors controlling disease, and especially Trichoderma is favored by researcher.Trichoderma (Trichoderma) fungi is widely present in soil and other substratess, and survival ability is strong, wide adaptability, is a kind of broad spectrum activity Antagonistic Fungi.Therefore, trichoderma is the most desired bio-control factors for being generally considered to replace a variety of chemical bactericides.For many years Come, people's study emphasis the bacterial strain heredity Gai Liang ﹑ resistance Ji ﹑ formulation development of Trichoderma and grinding for Biocontrol Mechanism etc. Study carefully work.Trichoderma has one to participate to several mechanism during antagonism pathogen, mainly includes antibiosis, and induction is anti- Property, the mechanism such as generation and hyperparasite of cell degradation enzyme.In addition, further including due to the auxin substance that Trichoderma generates to plant The growth-promoting functions etc. that object generates.In recent years, have many articles report successively certain kinds of the bacterium it is former to a variety of soil-borne diseases true Bacterium has antagonism, and the example that manufactured microbial inoculum is succeeded in greenhouse and Field information, is prevented and treated using trichoderma withered by standing Rhizoctonia (Rhizoctonia solani), pythium spp (Pythium spp), Sclerotium rolfsii (Sclerotium Rolfsii), the damping-off of larch Seedlings of cotton, Cortex Eucommiae, ginseng, Radix Notoginseng caused by sickle-like bacteria (Fusarium spp) etc. etc., and it is right Jasmine, the southern blight of peanut and capsicum, samping off of tomato etc. have obtained favorable effect in various small-scale tests, some of Type has been registered as microbial bactericide.The Trichoderma harzianum T-22 in the U.S. and the Trichoderma harzianum T39 of Israel are registered Registration, with the continuous development of modern biotechnology, the biocontrol effect and stability of Trichoderma have significant raising, due to it It is free from environmental pollution, thus have broader practice prospect.
Summary of the invention
The object of the invention first is that agricultural production and agricultural product quality and safety there are aiming at the problem that and the prior art not Foot, provides one plant of koning trichoderma bacterium (Trichoderoma koningii) TG-72 for having favorable effect to Aspergillus flavus.
It is a further object of the present invention to provide by above-mentioned koning trichoderma bacterium (Trichoderoma koningii) TG-72 system Standby microbial inoculum, preparation method and its application in Aspergillus flavus biological control.
For achieving the above object, the technical solution adopted by the present invention are as follows:
Koning trichoderma bacterium (Trichoderoma koningii) TG-72, is preserved in Chinese Typical Representative on January 7th, 2016 Culture collection (CCTCC), deposit number are CCTCC NO:M 2016015, classification naming Trichoderoma koningii TG-72.Koning trichoderma bacterium TG-72 is to separate to obtain from the peanut pod of the south china town Ba Bu, through indoor face-off The antibacterial removing toxic substances experiment of antagonistic experiment, living body and field controling test discovery, the bacterial strain bacteriostasis is stronger, and antibacterial detoxifying effect is good And stablize, it is easy culture, it is pollution-free, it is environmentally safe.
The separation of koning trichoderma bacterium TG-72 bacterial strain and screening technique are as follows:
(1) sample acquires: acquisition sample is peanut pod or Peanut Root System, and place is south china, Jiangxi camphor tree, Hebei The peanut cultivations area such as Tangshan and Henan Xinxiang.The topsoil for pushing field 3-5cm aside with soil sampler material, by soil together with peanut Pod and root system are dug out together, are installed with polyethylene plastic bag, and laboratory separation is taken back.
(2) separation method: the root system for adhering to soil slightly being air-dried, root system is patted, and the soil for making to be adhered to above falls off, and uses Aseptic water washing is clean, and peanut pod or root system are ground with mortar, is diluted with dilution gradient method, draws 0.1mL respectively Dilution instills on TSM culture medium flat plate, is coated with uniformly, is placed in 25-28 DEG C of constant incubator with the L shape spreading rod of sterilizing Culture 3-4 days.Picking form is transferred on PDA plate like the single colonie of Trichoderma and carries out purifying culture, and mirror mirror is tentatively regarded as It numbers, and is saved on the inclined-plane PDA after Trichoderma, further progress is in vitro, living body antagonistic experiment and field trial screening obtain.
Trichoderma culture medium semi-selective (TSM culture medium): MgSO4(7H20) 0.2g, K2HPO40.9g, KCl 0.15g, NH4NO31.0g, glucose 3.0g, rose-red 0.15g, 60% 0.3 gram of fenaminosulf wettable powder, PCNB0.2g, agar 20g, Distilled water 950ml.20 DEG C of 121min sterilizing is spare.
Biocontrol microorganisms TG-72 is koning trichoderma through Molecular Identification, and ITS gene order is shown in specification nucleotide sequence and amino Acid sequence SEQ.ID.NO.1.
Morphological feature:
20 DEG C of 5d colony diameter 9.0cm in PDA culture medium, quickly, spider's thread shape to ulotrichy is white, scattered for the speed of growth Due to producing spore surface in green under light, the back side is colourless, and the later period is since sporulation quantity ambassador's bacterium colony is in slightly green；Tree-shaped point of multi-branched Raw sporophore forms quite loose flora；Major branch is 4-5 μm wide, and side shoot is more, forms pyramid；The raw bottle stalk in side is 5 reachable, at Quasi- colyliform arranges or individually along small side shoot irregular alignment；The raw bottle metulae portion in side slightly hangs contracting, and middle part is expanded, gradually narrow at bottle upwards Stalk, 5-7 × 3-3.5 μm；Basidixed and atypical bottle of stalk is relatively long and very thin, and 13-18 × 2.5-3.5 μm；Bottle stalk mostly with Wide-angle on its carrier it is raw, it is sometimes slightly curved to top；Phialospore be in spheric conidium head, conidium it is subsphaeroidal or Obovate, wall is smooth, light green, and 2.8-3.2 × 2.5-2.8 μm.As depicted in figs. 1 and 2.
The biological activity determination of koning trichoderma TG-72 bacterial strain:
(1) inhibiting effect of the trichoderma TG-72 bacterial strain to pathogen: it is respectively connected at 3cm on PDA plate culture medium The trichoderma bacteria cake and cause of disease bacteria cake prepared is opposed culture of standing erect, each for trying the bacteria cake of bacterium as control to be individually inoculated with, and is placed in culture 25 DEG C of dark culturings of case.Every processing sets 3 repetitions.Colony diameter is measured after 3d, calculates suppression of the strains tested to growth of pathogenic bacteria Rate processed.Table 1 indicate applied by the preferable biocontrol microorganisms of effect to the inhibiting rate of Aspergillus flavus, in the 325 plants of bacterial strains tested, bacterium Strain TG-72 bacterial strain is most strong to the inhibiting effect of Aspergillus flavus, and inhibiting rate reaches 55.5%, followed by the 49.2 of TG-22.
Inhibiting rate (%) of 7 bacterial strains such as 1 trichoderma TG-72 of table to aspergillus flavus bacterium
Note: the experimental data in table is duplicate average value three times.
(2) the indoor living biocontrol effect experiment of trichoderma TG-72 bacterial strain
1. the preparation of antagonism inoculum: the trichoderma strain in test tube slant culture medium being inoculated into PDA in eggplant bottle and is cultivated On base, under the conditions of 25-28 DEG C, cultivate 3-4 days in the incubator.Reesei spores in eggplant bottle are swept away with sterile water, are inoculated with Into medium-sized fermentor.Fermentation process holding dissolved oxygen concentration 20% or so, 28-30 DEG C of temperature, mixing speed 200-250r/ Min, ventilatory capacity 10-15L/min.Fermentation liquid is inoculated in solid medium by the inoculum concentration of volume 10%, inoculation finishes After be transferred in culturing room cultivate.Room temperature control is cultivated ± 1 DEG C of (28-30), culture medium temperature is controlled in (25-28) ± 1 DEG C, relative air humidity control is 5-7 days in 95-98%, culture medium thickness 5-7cm, incubation time.It, will after culture Culture natural air drying obtains the fermenting agent of antagonistic strain.
2. the Vivo Studies on Screening of Antagonistic Fungi: it is 1.0 × 10 that above-mentioned antagonism fermented liquid, which is diluted to spore ultimate density,6Spore Sub- g-17 days eugonic Aspergillus flavus bacteria suspensions of spore suspension and culture (spore ultimate density is 1.0 × 106Spore ML-1 it) is soaked respectively flower bulb non-hibernating eggs 30min, shady place is put into aseptic flat board after being done, and 20 peanuts of every plate are put into illumination box Cultivate 10d.Incubator condition of culture are as follows: 28 DEG C of temperature, humidity 70-80%, illumination is 12h dark/12h illumination.Not soak bacterium Liquid is blank control, and every processing sets 3 repetitions.Table 3 points out bacterial strain TG-72 inhibiting rate and presses down malicious rate highest, respectively reaches 85.0% and 85.5%, which has further Potential as a researcher (table 2 and Fig. 3).Press down malicious rate (%)=(control toxin amount-place Manage toxin amount)/control toxin amount × 100%；Inhibiting rate (%)=(control morbidity number-processing morbidity)/control morbidity number × 100%.
7 plants of biocontrol microorganisms are calculated to the inhibiting rate of Aspergillus flavus and endotoxin contamination and press down malicious rate (table 2 and Fig. 3).
27 plants of Antagonistic Fungis of table are to the control efficiency of Aspergillus flavus and the malicious rate of suppression
Note: data are duplicate average value three times
A kind of microbial organic fertilizer microbial inoculum of prevention and treatment Aspergillus flavus produced with above-mentioned koning trichoderma TG-72 bacterial strain is provided.
Preparation method: koning trichoderma bacterium TG-72 solid fermentation object and manioc waste and feces of livestock and poultry mixture high temperature is decomposed Material carries out solid fermentation, adjusts water content, and fermentation carries out stirring two days later, carries out turning daily later, can ferment knot for 5-7 days Beam, then progress low temperature drying (≤60 DEG C) makes its water content≤30% that can obtain packed products solid fermentation microorganism organic Fertilizer, antagonism bacterial content is up to 5 × 10 in solid fermentation microbial organic fertilizer8Cfu/g or more, content of organic matter 38-45wt%.
According to the above scheme, the preparation method of koning trichoderma bacteria solid fermentation object:
(1) spore suspension prepares: by the spore of koning trichoderma TG-72 or mycelium inoculation in PDA culture medium, 25-28 DEG C Culture 7 days, grows up to green to spore；
(2) eggplant bottle culture: trichoderma strain spore suspension is inoculated into eggplant bottle in PDA culture medium, in 25-28 DEG C of condition Under, it cultivates 3-4 days in the incubator；
(3) seed flask culture: the reesei spores in eggplant bottle are swept away with sterile water, is inoculated into and is placed with fluid nutrient medium In fermentor, it is 15-25% that fermentation process, which keeps dissolved oxygen concentration, 28-30 DEG C of temperature, mixing speed 200-250r/min, is led to Tolerance is 10-15L/min.
(4) solid fermentation culture；Liquid spawn is inoculated in solid medium by 10% inoculum concentration by volume, inoculation After be transferred in culturing room and cultivate, ± 1 DEG C of (28-30), culture medium temperature is controlled at (25-28) for culture room temperature control ± 1 DEG C, relative air humidity control is 5-7 days, after culture in 95-98%, culture medium thickness 5-7cm, incubation time, By culture natural air drying, finished product water content is controlled in 7-10%, obtains koning trichoderma bacteria solid fermentation object.
According to the above scheme: PDA culture medium: potato 200g, glucose 15g, agar 20g, distilled water 1000ml.
According to the above scheme: fluid nutrient medium: wheat bran 2.2wt%, glucose 1.0wt%, magnesium sulfate 0.45wt%, di(2-ethylhexyl)phosphate Hydrogen potassium 0.35wt%, sterilize under 0.3wt%, pH6.5-6.8,121 DEG C of wet heat conditions of calcium chloride 30min,.
According to the above scheme: wheat bran is counted in solid medium in mass ratio: wheat straw powder=7:3, water content 70wt%, PH6.5,121 DEG C of sterilizing 30min.
According to the above scheme, koning trichoderma bacterium solid hair is added in manioc waste per ton and the decomposed material of feces of livestock and poultry mixture high temperature 15-30 kilograms of ferment object.
3. the antibacterial removing toxic substances rate in trichoderma TG-72 bacterial strain bio-bacterial manure field and growth-promoting functions
Field trial cell Random Design, 3 repetitions, the area of each cell are 15m × 3m.By biological prevention and control agent with per acre It 40-60 kilograms, is applied when peanut seeding with fertilizer, chemical prevention and control method (70% thiophanate methyl wettable powder is set 0.5g/) and control, the same biological prevention and control agent of the method for administration of pesticide.Every 5 points of processing random searching after harvesting peanut, every 10 Strain.Table 3 points out bacterial strain TG-72 growth-promoting rate and presses down malicious rate highest, respectively reaches 12.5% and 90.5%, which, which has, further grinds The potentiality studied carefully, followed by TG11-1 press down malicious rate and reach 60.5% (table 3).Press down malicious rate (%)=(control toxin amount-processing toxin Amount)/control toxin amount × 100%；Growth-promoting rate (%)=(control plant height-processing plant height)/control plant height × 100%.
3 Antagonistic Fungi of table produces malicious inhibiting rate to Aspergillus flavus and to peanut growth-promoting effect
A kind of microbial organic fertilizer of Aspergillus flavus on the above-mentioned prevention and treatment peanut Aspergillus flavus on field control peanut is provided In application, application method are as follows: by prevent and treat peanut on Aspergillus flavus microbial organic fertilizer preparation with 40-60 kilograms per acre, in It is applied when peanut seeding with fertilizer.
Detailed description of the invention
Fig. 1 is the PDA plate culture form of trichoderma TG-72.
Fig. 2 is the conidiophore and conidium of trichoderma TG-72.
Fig. 3 is inhibiting effect of the trichoderma TG-72 to aspergillus flavus germ on peanut.
Fig. 4 is inhibiting effect of the trichoderma TG-72 metabolin to Aspergillus flavus on plate.
Specific embodiment
Embodiment 1: the solid fermentation culture of trichoderma TG-72 bacterial strain is weight percentage below.
The trichoderma TG-72 bacterial strain being related in the present invention, deposit number are CCTCC NO:M 2016015, and classification naming is Trichoderoma koningii TG-72.It is to separate to obtain from south china's Peanut Root System, real through indoor face-off antagonism It tests, living body suppression poison experiment and the discovery of crop field toxin inhibitory test, the bacterial strain is antibacterial relatively strong, and antibacterial detoxifying effect is good and stablizes, and is easy to train It supports, it is pollution-free, it is environmentally safe.
The solid fermentation culture of trichoderma TG-72 bacterial strain and preparation preparation are as follows:
(1) slant strains: by the spore of trichoderma TG-72 bacterial strain or mycelium inoculation in PDA culture medium, 25-28 DEG C of culture 5 It.
(2) eggplant bottle culture: the trichoderma strain in test tube slant culture medium is inoculated into eggplant bottle in PDA culture medium, in 25- Under the conditions of 28 DEG C, cultivate 3-4 days in the incubator.
(3) seed flask culture: seed culture medium, wheat bran 2.2%, glucose 1.0%, magnesium sulfate 0.45%, phosphoric acid are used Potassium dihydrogen 0.35%, sterilize under calcium chloride 0.3%, PH6.5-6.8,121 DEG C wet heat condition 30min, by the trichoderma spore in eggplant bottle Son is swept away with sterile water, is inoculated into medium-sized fermentor.Fermentation process holding dissolved oxygen concentration 20% or so, 28-30 DEG C of temperature, Mixing speed 200-250r/min, ventilatory capacity 10-15L/min.
(4) solid fermentation culture: wheat bran is counted in mass ratio in solid medium: wheat straw powder=7:3, water content 70%, PH6.5,121 DEG C of sterilizing 30min.Liquid spawn is inoculated in solid medium by 10% inoculum concentration by volume, has been inoculated with It is transferred in culturing room and cultivates after finishing.Room temperature control is cultivated ± 1 DEG C of (28-30), culture medium temperature control (25-28) ± 1 DEG C, relative air humidity control is 5-7 days in 95-98%, culture medium thickness 5-7cm, incubation time.It, will after culture Culture natural air drying, finished product water content are controlled in 7-10%, obtain koning trichoderma bacteria solid fermentation object.
5) manioc waste and feces of livestock and poultry mixed-stacking are fermented 10-15 days and obtains manioc waste and feces of livestock and poultry mixture high temperature Decomposed material, manioc waste and the decomposed material of feces of livestock and poultry mixture high temperature: germination index >=98%, content of organic matter 35-40wt% contain Koning trichoderma bacteria solid fermentation object is added in water 15-20wt%, manioc waste per ton and the decomposed material of feces of livestock and poultry mixture high temperature 20 kilograms, adjust water content, fermentation carry out stirring two days later, later daily carry out turning, 5-7 days can fermentation ends, then Carrying out low temperature drying (≤60 DEG C) makes its water content≤30% that packed products solid fermentation microbial organic fertilizer, solid can be obtained Antagonism bacterial content is up to 5 × 10 in fermentative microorganism organic fertilizer8Cfu/g or more, content of organic matter 38-45%.
Embodiment 2
The measurement of trichoderma TG-72 Metabolite bacteriostasis rate
The preparation of biocontrol microorganisms metabolite
After biocontrol microorganisms TG-72 is activated on the inclined-plane PDA, take a ring in PD culture solution, 100mL/300mL liquid amount, 30 DEG C, after 160r/min shaken cultivation 72h, be prepared into following treatment fluid: supernatant: culture solution is centrifuged at 12000r/min 15min takes supernatant, obtains sterile supernatant after being filtered with 0.22 μm of biofilter.
Bacteria suspension: culture solution is centrifuged 15min at 12000r/min, abandons supernatant, is centrifuged, is added again with sterile water wash 3 times Enter sterile water.
Protein crude extract: culture solution 12000r/min at 4 DEG C is abandoned from 15min to be precipitated, the reinforcing body sulfuric acid in supernatant To 70% saturation degree, 4 DEG C stand overnight ammonium, and 10000r/min is centrifuged 20min at 4 DEG C, abandon supernatant, precipitate 1/25 volume 10mmol/L, pH7.0 phosphate buffer suspend, and are then removed by filtration bacterium that may be present with 0.22 μm of biofilter.
The influence that biocontrol microorganisms metabolite grows Aspergillus flavus mycelia
Measuring method: Aspergillus flavus bacteria suspension 1 × 10 is taken6Cfu/ml 5mL is put into temperature and drops to 45 DEG C or so PDA triangles In bottle (100mL/350mL), after rocking 2min, uniformly pour into culture dish.It is beaten with punch is equidistant around PDA plate 6 holes are separately added into 3mL supernatant, filtered fluid, freezing supernatant, freezing and filtering liquid and protein crude extract, culture with liquid-transfering gun Liquid, 30 DEG C of cultures detect the radius of each processing inhibition zone, three repetitions of each processing for 5 days when CK covers with plate.Meanwhile it studying Thermal stability experiment of the biocontrol microorganisms supernatant at 40,50,60,70,80,100 DEG C, thermal stability experiment are soaked in water-bath 1h is heated, handles 20min at a temperature of 121 DEG C.Experimental result shows, the bacteriostatic diameter of bacterial strain TG-72 culture solution up to 24.5mm, Followed by supernatant, freeze the 6.5mm of clear 12.0mm and protein crude extract, rejection ability is stronger.Culture solution, supernatant, jelly Supernatant is shown in Fig. 4 to inhibiting effect of the Aspergillus flavus on plate.Meanwhile bacterial strain TG-72 bacterial strain supernatant is more than 60 DEG C and loses To the rejection ability of Aspergillus flavus, the metabolite for illustrating that the bacterial strain generates is protein-based or lipopeptid substance.Experimental result Such as table 4 and table 5:
The inhibiting effect that 4 biocontrol microorganisms metabolite of table grows Aspergillus flavus mycelia
Table 5 is heat-treated the influence to biocontrol microorganisms metabolite bacteriostasis
Embodiment 3
The root colonization of trichoderma TG-72 bacterial strain is tested
Peanut 8252 selects the consistent germination kind sowing of growing way through indoor vernalization, and 10, every basin repeats five times.To peanut Start to connect bacterium after the flattening of seedling two panels cotyledon.The spore suspension concentration of trichoderma TG-72 bacterial strain is 2 × 106/ mL, spore is hanged Supernatant liquid syringe injects rhizosphere soil, and every cave 15mL, clear water is as control.After 4 weeks, planted with TSM culture based assays potato Trichoderma TG-72 bacterial strain in strain rhizosphere soil, in this, as the index of trichoderma Growth of Rhizosphere Fungi colonization ability.Not to be inoculated with when counting The peanut plant rhizosphere soil of Trichoderma is as control.It the results are shown in Table 6, display, no matter bacterial strain TG-72 is individually inoculated with or and Huang Qu Mould mixed bacteria, 7 days and 14 days survival rates are unobvious, and since 28 days, the soil bacteria containing amount for being only inoculated with biocontrol microorganisms was significant Higher than the combined inoculation with aspergillus flavus.
6 trichoderma TG-72 of table is in peanut plant rhizospere competition (× 10x/g)
Note: the experimental data in table is duplicate average value three times.

Claims (10)

1. koning trichoderma bacterium (Trichoderoma koningii) TG-72, it is characterised in that: it is preserved on January 7th, 2016 China typical culture collection center, deposit number are CCTCC NO:M 2016015.
2. a kind of microbial organic fertilizer system for preventing and treating Aspergillus flavus made of koning trichoderma bacterium TG-72 described in claim 1 Agent.
3. microbial organic fertilizer preparation according to claim 2, it is characterised in that: short of money in the microbial organic fertilizer preparation Antibacterial content is 5 × 108Cfu/g or more, content of organic matter 35-40wt%.
4. the production method of microbial organic fertilizer preparation as claimed in claim 3, it is characterised in that: by koning trichoderma bacterium TG-72 Solid fermentation object and the decomposed material of manioc waste and feces of livestock and poultry mixture high temperature carry out solid fermentation, adjust water content, ferment two days After carry out stirring, later daily carry out turning, 5-7 days fermentation ends, then progress low temperature drying obtain its water content≤30% Packed products solid fermentation microbial organic fertilizer, antagonism bacterial content is up to 5 × 10 in solid fermentation microbial organic fertilizer8Cfu/g with On, content of organic matter 38-45wt%.
5. the production method of microbial organic fertilizer preparation according to claim 4, it is characterised in that: manioc waste and animal dung Just the decomposed material of mixture high temperature: germination index >=98%, content of organic matter 35-40wt%, water content 15-20wt%.
6. the production method of microbial organic fertilizer preparation according to claim 4, it is characterised in that: koning trichoderma bacterium TG- The preparation method of 72 solid fermentation objects:
(1) spore suspension prepares: by the spore of koning trichoderma bacterium TG-72 or mycelium inoculation in PDA culture medium, 25-28 DEG C Culture 7 days, grows up to green to spore；
(2) eggplant bottle culture: trichoderma strain spore suspension is inoculated into eggplant bottle in PDA culture medium, under the conditions of 25-28 DEG C, It cultivates 3-4 days in the incubator；
(3) seed flask culture: the reesei spores in eggplant bottle are swept away with sterile water, are inoculated into the fermentation for being placed with fluid nutrient medium In tank, it is 15-25% that fermentation process, which keeps dissolved oxygen concentration, and 28-30 DEG C of temperature, mixing speed 200-250r/min, ventilatory capacity is 10-15L/min；
(4) solid fermentation culture；Liquid spawn is inoculated in solid medium by 10% inoculum concentration by volume, and inoculation finishes After be transferred in culturing room and cultivate, ± 1 DEG C of (28-30), culture medium temperature is controlled in (25-28) ± 1 for culture room temperature control DEG C, relative air humidity control is 5-7 days in 95-98%, culture medium thickness 5-7cm, incubation time, after culture, will be trained Object natural air drying is supported, finished product water content is controlled in 7-10%, obtains koning trichoderma bacteria solid fermentation object.
7. the production method of microbial organic fertilizer preparation according to claim 6, it is characterised in that: PDA culture medium: Ma Ling 200 g of potato, 15 g of glucose, 20 g of agar, 1000 ml of distilled water；
Fluid nutrient medium: wheat bran 2.2wt%, glucose 1.0wt%, magnesium sulfate 0.45wt%, potassium dihydrogen phosphate 0.35wt%, calcium chloride Sterilize 30min under 0.3wt%, pH6.5-6.8,121 DEG C of wet heat conditions；
Wheat bran is counted in solid medium in mass ratio: wheat straw powder=7:3,70wt%, pH6.5,121 DEG C of sterilizing 30min of water content.
8. the production method of microbial organic fertilizer preparation according to claim 4, it is characterised in that: manioc waste per ton and poultry 15-30 kilograms of object of koning trichoderma bacteria solid fermentation is added in the decomposed material of poultry manure mixture high temperature.
9. a kind of application of koning trichoderma bacterium TG-72 described in claim 1 in Aspergillus flavus biological control.
10. microbial organic fertilizer described in a kind of any one of claim 2-8 is on field control peanut in Aspergillus flavus Using, which is characterized in that application method are as follows: by microbial organic fertilizer preparation with 40-60 kilograms per acre in peanut seeding Shi Suifei Material applies.
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