CN105695342A - Trichoderoma koningii TC-72 and application of trichoderoma koningii TC-72 to biological control of aspergillus flavus - Google Patents
Trichoderoma koningii TC-72 and application of trichoderoma koningii TC-72 to biological control of aspergillus flavus Download PDFInfo
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Abstract
The invention relates to trichoderoma koningii TC-72 and application of the trichoderoma koningii TC-72 to biological control of aspergillus flavus. The trichoderoma koningii TC-72 is preserved in the China Center For Type Culture Collection on January 7th, 2016, and the preservation number is CCTCC NO:M 2016015. The trichoderoma koningii TC-72 is obtained by being separated out of peanut pods of Babu Town, Hezhou City, Guangxi Province. It is found through indoor antagonistic experiments, living body bacteriostatic detoxification experiments and field controlling experiments that a bacterial strain is high in bacteriostatic ability, good and stable in bacteriostatic detoxification effect, easy to culture, free of pollution and safe to environment. After bacterial manure is applied to soil, antagonistic bacteria can be fast reproduced on the rhizosphere of crops and survive for a long time, a dominant bacterial community is formed, and harm of aspergillus flavus to peanuts can be effectively controlled.
Description
Technical field
The present invention relates to useful fungus application, be specifically related to trichoderma pseudokiningii bacterium TC-72 and the application in Aspergillus flavus Biological control thereof。
Background technology
The endotoxin contamination caused by aspergillus fungi (particularly Aspergillus flavus) is referred to as aspergillosis endotoxin contamination, and host range is wide。Aspergillosis particularly Aspergillus flavus brings serious harm to the safety in production of Semen arachidis hypogaeae, Semen Maydis, Oryza sativa L. and Soybean and Other Crops, and the economic loss caused every year is up to tens billion of dollars。Aspergillus fungi particularly Aspergillus flavus can produce the mycotoxin that toxicity is extremely strong。Aflatoxin is a class carcinogenic, the hypertoxicity mycotoxin by force extensively polluting agricultural product, including B, G and M race, wherein B1 is the most general and toxicity is the strongest, for potassium cyanide 10 times, 68 times of arsenicum, carcinogenicity is 10000 times of Isotox, and human or animal can cause that body disease is even dead after eating the food by endotoxin contamination or feedstuff, serious threat consumer health and life security, restriction agricultural industry development and international trade。Therefore, the prevention and control of Aspergillus flavus and endotoxin contamination in the agricultural product such as China's Semen arachidis hypogaeae, Semen Maydis and Oryza sativa L. are strengthened very urgent。
At present, in the preventing and treating to Aspergillus flavus, chemical prevention occupies consequence。Chemical prevention not only cost is high, and easy contaminated environment, and while prevention and control pathogen, chemical agent is also easily produced drug resistance even Drug resistance by pathogen。Therefore, strengthening Aspergillus flavus biology prevention and control, be increasingly becoming focus and focus that people pay close attention to, the raising of China's agricultural industry development and agricultural product quality is had great importance by this。
Domestic utilize in bio-control factors controlling disease, particularly the favor of trichoderma extremely research worker。Trichoderma (Trichoderma) fungus is widely present in soil and other base thing, and survival ability is strong, wide adaptability, is a kind of broad spectrum activity Antagonistic Fungi。Therefore, Trichoderma spp. is the most desired bio-control factors being generally considered to replace multiple chemical bactericide。For many years, the research work of the aspect such as bacterial strain genetic improvement resistance mechanism formulation development and Biocontrol Mechanism of people's study emphasis trichoderma。Trichoderma spp. has one to participate to several mechanism in the process of antagonism pathogen, mainly includes the mechanism such as antibiosis, induction of resistance, the generation of cell degradation enzyme and superparasitism。Additionally, the growth-promoting functions etc. also including the auxin substance because trichoderma produces and plant being produced。In recent years, multiple soil-borne disease fungal pathogens is had antagonism by some kind that existing many articles report this bacterium successively, and the microbial inoculum made obtains successful example in greenhouse and Field information, Trichoderma spp. is utilized to prevent and treat by Rhizoctonia solani Kuhn (Rhizoctoniasolani), pythium spp (Pythiumspp), Sclerotium rolfsii (Sclerotiumrolfsii), the Cotton Gossypii that Fusarium spp. (Fusariumspp) etc. causes, the Cortex Eucommiae, Radix Ginseng, the damping-off of larch Seedlings of Radix Notoginseng etc., and to jasmine, the southern blight of Semen arachidis hypogaeae and Fructus Capsici, the damping ofies of Fructus Lycopersici esculenti etc. have obtained favorable effect in various small-scale tests, some of them kind has been registered as microbial bactericide。The Trichoderma harzianum T-22 of the U.S. and the Trichoderma harzianum T39 of Israel obtains registration, development along with modern biotechnology, the biocontrol effect of trichoderma and stability have had significant raising, owing to it is free from environmental pollution, thus have broader practice prospect。
Summary of the invention
One of the object of the invention is for agricultural production and agricultural product quality and safety Problems existing and the deficiencies in the prior art, it is provided that Aspergillus flavus is had trichoderma pseudokiningii bacterium (Trichoderomakoningii) TC-72 of favorable effect by a strain。
It is a further object of the present invention to provide microbial inoculum, preparation method and the application in Aspergillus flavus Biological control thereof prepared by above-mentioned trichoderma pseudokiningii bacterium (Trichoderomakoningii) TC-72。
For achieving the above object, the technical solution used in the present invention is:
Trichoderma pseudokiningii bacterium (Trichoderomakoningii) TC-72, it is preserved in China typical culture collection center (CCTCC) on January 7th, 2016, preserving number is CCTCCNO:M2016015, and Classification And Nomenclature is TrichoderomakoningiiTG-72。Trichoderma pseudokiningii bacterium TC-72 separates to obtain from the peanut pod of Ba Bu town, south china, finds through indoor face-off antagonistic experiment, the antibacterial removing toxic substances experiment of live body and field controling test, and this bacterial strain bacteriostasis is stronger, antibacterial detoxifying effect is good and stable, easily cultivate, pollution-free, environmentally safe。
Separation and the screening technique of trichoderma pseudokiningii bacterium TC-72 bacterial strain are as follows:
(1) sample collecting: collected specimens is peanut pod or Peanut Root System, place is the peanut cultivation areas such as south china, Jiangxi Lignum cinnamomi camphorae, Tangshan and Xinxiang, Henan。Push the topsoil of field 3-5cm with the equipment that fetches earth aside, the soil pod together with Semen arachidis hypogaeae and root system are dug out, installs with polyethylene plastic bag, take back laboratory and separate。
(2) separation method: by slightly air-dry for the root system of adhesion soil, pat root system, the superincumbent soil of adhesion is made to come off, clean with aseptic water washing, with mortar, peanut pod or root system are ground, be diluted by dilution gradient method, draw 0.1mL diluent respectively and instill on TSM culture medium flat plate, it is coated with uniformly with the L-shaped spreading rod of sterilizing, is placed in the constant incubator of 25-28 DEG C and cultivates 3-4 days。Picking form is transferred on PDA plate is purified cultivation like single bacterium colony of trichoderma, and mirror mirror is numbered after tentatively regarding as trichoderma, and is saved on PDA inclined-plane, carries out in vitro, live body antagonistic experiment further and field test screening obtains。
Trichoderma semi-selection culture medium (TSM culture medium): MgSO4(7H20) 0.2g, K2HPO40.9g, KCl0.15g, NH4NO31.0g, glucose 3.0g, rose-red 0.15g, 60% fenaminosulf wettable powder 0.3 gram, PCNB0.2g, agar 20g, distilled water 950ml。121min sterilizing 20 DEG C is standby。
Biocontrol microorganisms TC-72 is trichoderma pseudokiningii through Molecular Identification, and ITS gene order is shown in description nucleotide sequence and aminoacid 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 is to ulotrichy, white, and owing to producing spore surface in green under scattered light, the back side is colourless for the speed of growth, and the later stage is owing to sporulation quantity ambassador's bacterium colony is in slightly green；The tree-shaped conidiophore of multi-branched forms quite loose flora；Major branch width 4-5 μm, side shoot is many, forms pyramid；The raw bottle in side obstructs up to 5, becomes to intend colyliform arrangement or single along little side shoot irregular alignment；Slightly hanging contracting in raw bottle metulae portion, side, middle part is expanded, and upwards gradually narrow one-tenth bottle obstructs, 5-7 × 3-3.5 μm；Push up the relatively long and very thin with atypical bottle of stalk of life, 13-18 × 2.5-3.5 μm；Bottle stalk mostly with wide-angle on its carrier raw, sometimes slightly curved to top；Phialospore is spheric conidium head, and conidium is subsphaeroidal or obovate, and wall is smooth, light green, 2.8-3.2 × 2.5-2.8 μm。As depicted in figs. 1 and 2。
The biological activity determination of trichoderma pseudokiningii TC-72 bacterial strain:
(1) the Trichoderma spp. TC-72 bacterial strain inhibitory action to pathogen: be respectively connected to, at a distance of 3cm place, the trichoderma cake for preparing in PDA plate culture medium and opposite culture made by pathogen cake, individually to inoculate each bacterium cake for examination bacterium for comparison, is placed in 25 DEG C of dark culturing of incubator。Often process and set 3 repetitions。Measure colony diameter after 3d, calculate the strains tested suppression ratio to growth of pathogenic bacteria。Table 1 indicates the effect the applied better biocontrol microorganisms suppression ratio to Aspergillus flavus, and in the 325 strain bacterial strains tested, bacterial strain TG-72 bacterial strain is the strongest to the inhibitory action of Aspergillus flavus, it is suppressed that rate reaches 55.5%, next to that the 49.2 of TG-22。
7 bacterial strains suppression ratio (%) to aspergillus flavus bacterium such as table 1 Trichoderma spp. TC-72
Note: the experimental data in table is three meansigma methodss repeated。
(2) the indoor living biocontrol effect experiment of Trichoderma spp. TC-72 bacterial strain
1. the preparation of Antagonistic Fungi inoculum: be inoculated in eggplant bottle in PDA culture medium by the Trichoderma spp. strain in test tube slant culture medium, under 25-28 DEG C of condition, cultivates 3-4 days in incubator。Reesei spores sterilized water in eggplant bottle is swept away, is inoculated in medium-sized fermentation tank。Sweat keeps dissolved oxygen concentration about 20%, and temperature 28-30 DEG C, mixing speed 200-250r/min, ventilation is 10-15L/min。By volume fermentation liquid is inoculated in solid medium by the inoculum concentration of 10%, transfers to cultivation indoor cultivation after inoculation。Culturing room's temperature controls in (28-30) ± 1 DEG C, and culture medium temperature controls in (25-28) ± 1 DEG C, and relative air humidity controls at 95-98%, and culture medium thickness is 5-7cm, and incubation time is 5-7 days。After cultivation, by culture natural air drying, it is thus achieved that the fermenting agent of antagonistic strain。
2. the Vivo Studies on Screening of Antagonistic Fungi: it is 1.0 × 10 that above-mentioned Antagonistic Fungi fermentation liquid is diluted to spore ultimate density6Spore g-1Spore suspension and cultivate 7 days eugonic Aspergillus flavus bacteria suspensions (spore ultimate density is 1.0 × 106Spore mL-1) soak Semen arachidis hypogaeae kind 30min respectively, shady and cool place puts into aseptic flat board, 20 Semen arachidis hypogaeaes of every plate after being done, and puts into illumination box and cultivates 10d。Incubator condition of culture is: temperature 28 DEG C, humidity 70-80%, and illumination is 12h dark/12h illumination。Not soak bacterium solution for blank, often process and set 3 repetitions。Table 3 point out bacterial strain TG-72 suppression ratio and press down poison rate the highest, respectively reach 85.0% and 85.5%, this bacterial strain has further Potential as a researcher (table 2 and Fig. 3)。Press down poison rate (%)=(comparison toxin amount-process toxin amount)/comparison toxin amount × 100%；Suppression ratio (%)=(comparison morbidity number-process morbidity)/comparison morbidity number × 100%。
Calculate 7 strain biocontrol microorganisms and to the suppression ratio of Aspergillus flavus and endotoxin contamination and press down poison rate (table 2 and Fig. 3)。
Table 27 strain Antagonistic Fungi is to the prevention effect of Aspergillus flavus and presses down poison rate
Note: data are three meansigma methodss repeated
The microbial organic fertilizer microbial inoculum preventing and treating Aspergillus flavus that the above-mentioned trichoderma pseudokiningii TC-72 bacterial strain of a kind of use produces is provided。
Preparation method: the material that trichoderma pseudokiningii bacterium TC-72 solid fermentation thing and manioc waste and feces of livestock and poultry mixture high temperature become thoroughly decomposed carries out solid fermentation, regulate water content, fermentation carries out stirring two days later, later every day carries out turning, within 5-7 days, get final product fermentation ends, then carry out cold drying (≤60 DEG C) make its water content≤30% packed products solid fermentation microbial organic fertilizer, in solid fermentation microbial organic fertilizer, Antagonistic Fungi content reaches 5 × 108More than cfu/g, the content of organic matter is 38-45wt%。
By such scheme, the preparation method of trichoderma pseudokiningii bacteria solid fermentation thing:
(1) spore suspension prepares: by the spore of trichoderma pseudokiningii TC-72 or mycelium inoculation in PDA culture medium, cultivates 7 days, treats that spore grows up to green for 25-28 DEG C；
(2) eggplant bottle is cultivated: be inoculated in eggplant bottle in PDA culture medium by Trichoderma spp. strain spore suspension, under 25-28 DEG C of condition, cultivates 3-4 days in incubator；
(3) seed flask is cultivated: swept away by the reesei spores sterilized water in eggplant bottle, being inoculated in the fermentation tank being placed with fluid medium, sweat keeps dissolved oxygen concentration to be 15-25%, temperature 28-30 DEG C, mixing speed 200-250r/min, ventilation is 10-15L/min。
(4) solid fermentation is cultivated；By volume the inoculum concentration of 10% by liquid-spawn inoculation in solid medium, cultivation indoor cultivation is transferred to after inoculation, culturing room's temperature controls in (28-30) ± 1 DEG C, and culture medium temperature controls in (25-28) ± 1 DEG C, and relative air humidity controls at 95-98%, culture medium thickness is 5-7cm, incubation time is 5-7 days, after cultivation, by culture natural air drying, finished product water content control, at 7-10%, obtains trichoderma pseudokiningii bacteria solid fermentation thing。
By such scheme: PDA culture medium: Rhizoma Solani tuber osi 200g, glucose 15g, agar 20g, distilled water 1000ml。
By such scheme: fluid medium: Testa Tritici 2.2wt%, glucose 1.0wt%, magnesium sulfate 0.45wt%, potassium dihydrogen phosphate 0.35wt%, sterilizing 30min under calcium chloride 0.3wt%, pH6.5-6.8,121 DEG C wet heat condition,。
By such scheme: Testa Tritici in mass ratio in solid medium: wheat straw powder=7:3, water content is 70wt%, pH6.5,121 DEG C of sterilizing 30min。
By such scheme, manioc waste per ton and feces of livestock and poultry mixture high temperature become thoroughly decomposed in expecting and add trichoderma pseudokiningii bacteria solid fermentation thing 15-30 kilogram。
3. the antibacterial removing toxic substances rate in Trichoderma spp. TG-72 bacterial strain bio-bacterial manure field and growth-promoting functions
Field test community Random Design, 3 repetitions, the area of each community is 15m × 3m。By biological prevention and control agent with every mu 40-60 kilogram, apply with fertilizer when peanut seeding, chemical prevention and control method (70% thiophanate methyl wettable powder 0.5g/) and comparison, the same biological prevention and control agent of application process of pesticide are set。5 points of random searching, every 10 strains are often processed after harvesting peanut。Bacterial strain TG-72 growth-promoting rate pointed out by table 3 and to press down poison rate the highest, respectively reaches 12.5% and 90.5%, and this bacterial strain has further Potential as a researcher, next to that TG11-1, presses down poison rate and reaches 60.5% (table 3)。Press down poison rate (%)=(comparison toxin amount-process toxin amount)/comparison toxin amount × 100%；Growth-promoting rate (%)=(comparison plant height-process plant height)/comparison plant height × 100%。
Aspergillus flavus is produced poison suppression ratio and to Semen arachidis hypogaeae growth-promoting effect by table 3 Antagonistic Fungi
Microbial organic fertilizer application in Aspergillus flavus on field control Semen arachidis hypogaeae of Aspergillus flavus on a kind of above-mentioned preventing and treating Semen arachidis hypogaeae is provided, application process is: with every mu 40-60 kilogram, applied with fertilizer when peanut seeding by the microbial organic fertilizer preparation of Aspergillus flavus on preventing and treating Semen arachidis hypogaeae。
Accompanying drawing explanation
The PDA plate that Fig. 1 is Trichoderma spp. TC-72 cultivates form。
Fig. 2 is conidiophore and the conidium of Trichoderma spp. TC-72。
Fig. 3 is Trichoderma spp. TC-72 to aspergillus flavus pathogenic bacteria inhibitory action on Semen arachidis hypogaeae。
Fig. 4 is Trichoderma spp. TC-72 metabolite to Aspergillus flavus inhibitory action on flat board。
Detailed description of the invention
Embodiment 1: the solid fermentation of Trichoderma spp. TC-72 bacterial strain is cultivated, and is all weight percentage below。
The Trichoderma spp. TC-72 bacterial strain related in the present invention, preserving number is CCTCCNO:M2016015, and Classification And Nomenclature is TrichoderomakoningiiTG-72。It is to separate to obtain from south china's Peanut Root System, and through indoor face-off antagonistic experiment, live body presses down poison experiment and land for growing field crops toxin inhibitory test finds, this bacterial strain is antibacterial relatively strong, and antibacterial detoxifying effect is good and stable, it is easy to cultivate, pollution-free, environmentally safe。
The solid fermentation of Trichoderma spp. TC-72 bacterial strain is cultivated and preparation preparation is as follows:
(1) slant strains: by the spore of Trichoderma spp. TC-72 bacterial strain or mycelium inoculation in PDA culture medium, cultivates 5 days for 25-28 DEG C。
(2) eggplant bottle is cultivated: be inoculated in eggplant bottle in PDA culture medium by the Trichoderma spp. strain in test tube slant culture medium, under 25-28 DEG C of condition, cultivates 3-4 days in incubator。
(3) seed flask is cultivated: adopt seed culture medium, Testa Tritici 2.2%, glucose 1.0%, magnesium sulfate 0.45%, potassium dihydrogen phosphate 0.35%, calcium chloride 0.3%, PH6.5-6.8, sterilizing 30min under 121 DEG C of wet heat conditions, sweeps away the reesei spores sterilized water in eggplant bottle, is inoculated in medium-sized fermentation tank。Sweat keeps dissolved oxygen concentration about 20%, and temperature 28-30 DEG C, mixing speed 200-250r/min, ventilation is 10-15L/min。
(4) solid fermentation is cultivated: counting Testa Tritici in solid medium in mass ratio: wheat straw powder=7:3, water content is 70%, pH6.5,121 DEG C of sterilizing 30min。The inoculum concentration of 10% is by liquid-spawn inoculation in solid medium by volume, transfers to cultivation indoor cultivation after inoculation。Culturing room's temperature controls in (28-30) ± 1 DEG C, and culture medium temperature controls in (25-28) ± 1 DEG C, and relative air humidity controls at 95-98%, and culture medium thickness is 5-7cm, and incubation time is 5-7 days。After cultivation, by culture natural air drying, finished product water content control, at 7-10%, obtains trichoderma pseudokiningii bacteria solid fermentation thing。
5) fermentation of manioc waste and feces of livestock and poultry mixed-stacking obtains manioc waste and feces of livestock and poultry mixture high temperature for 10-15 days become thoroughly decomposed material, manioc waste and feces of livestock and poultry mixture high temperature become thoroughly decomposed material: germination index >=98%, content of organic matter 35-40wt%, water content 15-20wt%, manioc waste per ton and feces of livestock and poultry mixture high temperature become thoroughly decomposed in expecting and add trichoderma pseudokiningii bacteria solid fermentation thing 20 kilograms, regulate water content, fermentation carries out stirring two days later, later every day carries out turning, within 5-7 days, get final product fermentation ends, then carrying out cold drying (≤60 DEG C) makes its water content≤30% can obtain packed products solid fermentation microbial organic fertilizer, in solid fermentation microbial organic fertilizer, Antagonistic Fungi content reaches 5 × 108More than cfu/g, the content of organic matter is 38-45%。
Embodiment 2
The mensuration of Trichoderma spp. TG-72 Metabolite bacteriostasis rate
The preparation of biocontrol microorganisms metabolite
After biocontrol microorganisms TG-72 activates on PDA inclined-plane, take a ring in PD culture fluid, 100mL/300mL liquid amount, 30 DEG C, after 160r/min shaken cultivation 72h, prepare into following treatment fluid: supernatant: culture fluid is centrifugal 15min under 12000r/min, takes supernatant, obtains aseptic supernatant after filtering with 0.22 μm of biofilter。
Bacteria suspension: culture fluid is centrifugal 15min under 12000r/min, abandons supernatant, is centrifuged for 3 times by sterile water wash again, adds sterilized water。
Protein crude extract: culture fluid 12000r/min at 4 DEG C abandons precipitation from 15min, in supernatant, reinforcing body ammonium sulfate is to 70% saturation, 4 DEG C stand overnight, the centrifugal 20min of 10000r/min at 4 DEG C, abandon supernatant, precipitate and suspend with 1/25 volume 10mmol/L, pH7.0 phosphate buffer, be then removed by filtration antibacterial that may be present with 0.22 μm of biofilter。
The impact on Aspergillus flavus mycelial growth of the biocontrol microorganisms metabolite
Assay method: take Aspergillus flavus bacteria suspension 1 × 1065mL, puts into temperature and drops in about 45 DEG C PDA triangular flasks (100mL/350mL), after rocking 2min, pour into uniformly in culture dish。6 holes are made a call to card punch is equidistant around PDA plate, it is separately added into 3mL supernatant, filtrate, freezing supernatant, freezing and filtering liquid and protein crude extract, culture fluid with liquid-transfering gun, cultivate the radius detecting each process inhibition zone for 5 days when CK covers with flat board, each process three repetition for 30 DEG C。Meanwhile, have studied the heat stability experiment at 40,50,60,70,80,100 DEG C of the biocontrol microorganisms supernatant, heat stability experiment is leaching heating 1h in water-bath, processes 20min at 121 DEG C of temperature。Experimental result shows, the bacteriostatic diameter of bacterial strain TG-72 culture fluid is up to 24.5mm, next to that supernatant, freeze the 6.5mm of clear 12.0mm and protein crude extract, it is suppressed that ability is stronger。Culture fluid, supernatant, freeze the clear inhibitory action to Aspergillus flavus on flat board and see Fig. 4。Meanwhile, namely bacterial strain TG-72 bacterial strain supernatant loses the rejection ability to Aspergillus flavus more than 60 DEG C, illustrates that the metabolite that this bacterial strain produces is protein-based or fat peptide matters。Experimental result such as table 4 and table 5:
The table 4 biocontrol microorganisms metabolite inhibitory action to Aspergillus flavus mycelial growth
The impact on biocontrol microorganisms metabolite bacteriostasis of table 5 heat treatment
Embodiment 3
The root colonization experiment of Trichoderma spp. TC-72 bacterial strain
Semen arachidis hypogaeae 8252, through indoor accelerating germination, is selected the germination kind sowing that growing way is consistent, 10, every basin, is repeated five times。Start to connect bacterium after peanut seedling two panels cotyledon flattens。The spore suspension concentration of Trichoderma spp. TC-72 bacterial strain is 2 × 106/ mL, injects rhizosphere soil by spore suspension syringe, and every cave 15mL, clear water is as comparison。After 4 weeks, measure the Trichoderma spp. TC-72 bacterial strain in potato plant rhizosphere soil by TSM culture medium, in this, as the index of Trichoderma spp. Growth of Rhizosphere Fungi colonization ability。Not inoculate the peanut plant rhizosphere soil of trichoderma as comparison during counting。Result in Table 6, display, no matter bacterial strain TG-72 individually inoculate or with Aspergillus flavus mixed bacteria, the survival rate of 7 days and 14 days is inconspicuous, and from 28 days, only the soil bacteria containing amount of inoculation biocontrol microorganisms was significantly higher than the combined inoculation with Aspergillus flavus。
Table 6 Trichoderma spp. TC-72 is at peanut plant rhizospere competition (× 10x/g)
Note: the experimental data in table is three meansigma methodss repeated。
Claims (10)
1. trichoderma pseudokiningii bacterium TC-72, it is characterised in that: being preserved in China typical culture collection center (CCTCC) on January 7th, 2016, preserving number is CCTCCNO:M2016015。
2. the microbial organic fertilizer preparation preventing and treating Aspergillus flavus that the trichoderma pseudokiningii bacterium TC-72 described in claim 1 makes。
3. microbial organic fertilizer preparation according to claim 2, it is characterised in that: in described microbial organic fertilizer preparation, Antagonistic Fungi content is 5 × 108More than cfu/g, the content of organic matter is 35-40wt%。
4. the production method of the microbial organic fertilizer preparation described in claim 3, it is characterized in that: the material that trichoderma pseudokiningii bacterium TC-72 solid fermentation thing and manioc waste and feces of livestock and poultry mixture high temperature become thoroughly decomposed carries out solid fermentation, regulate water content, fermentation carries out stirring two days later, later every day carries out turning, 5-7 days fermentation ends, then carry out cold drying and make its water content≤30% obtain packed products solid fermentation microbial organic fertilizer, and in solid fermentation microbial organic fertilizer, Antagonistic Fungi content reaches 5 × 108More than cfu/g, the content of organic matter is 38-45wt%。
5. the production method of microbial organic fertilizer preparation according to claim 3, it is characterised in that: manioc waste and feces of livestock and poultry mixture high temperature become thoroughly decomposed material: 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 3, it is characterised in that: the preparation method of trichoderma pseudokiningii bacterium TC-72 solid fermentation thing:
(1) spore suspension prepares: by the spore of trichoderma pseudokiningii TC-72 or mycelium inoculation in PDA culture medium, cultivates 7 days, treats that spore grows up to green for 25-28 DEG C；
(2) eggplant bottle is cultivated: be inoculated in eggplant bottle in PDA culture medium by Trichoderma spp. strain spore suspension, under 25-28 DEG C of condition, cultivates 3-4 days in incubator；
(3) seed flask is cultivated: swept away by the reesei spores sterilized water in eggplant bottle, being inoculated in the fermentation tank being placed with fluid medium, sweat keeps dissolved oxygen concentration to be 15-25%, temperature 28-30 DEG C, mixing speed 200-250r/min, ventilation is 10-15L/min。
(4) solid fermentation is cultivated；By volume the inoculum concentration of 10% by liquid-spawn inoculation in solid medium, cultivation indoor cultivation is transferred to after inoculation, culturing room's temperature controls in (28-30) ± 1 DEG C, and culture medium temperature controls in (25-28) ± 1 DEG C, and relative air humidity controls at 95-98%, culture medium thickness is 5-7cm, incubation time is 5-7 days, after cultivation, by culture natural air drying, finished product water content control, at 7-10%, obtains trichoderma pseudokiningii bacteria solid fermentation thing。
7. the production method of microbial organic fertilizer preparation according to claim 6, it is characterised in that: PDA culture medium: Rhizoma Solani tuber osi 200g, glucose 15g, agar 20g, distilled water 1000ml；
Fluid medium: Testa Tritici 2.2wt%, glucose 1.0wt%, magnesium sulfate 0.45wt%, potassium dihydrogen phosphate 0.35wt%, sterilizing 30min under calcium chloride 0.3wt%, pH6.5-6.8,121 DEG C wet heat condition；
Counting Testa Tritici in solid medium in mass ratio: wheat straw powder=7:3, water content is 70wt%, pH6.5,121 DEG C of sterilizing 30min。
8. the production method of microbial organic fertilizer preparation according to claim 3, it is characterised in that: manioc waste per ton and feces of livestock and poultry mixture high temperature become thoroughly decomposed in expecting and add trichoderma pseudokiningii bacteria solid fermentation thing 15-30 kilogram。
9. the application in Aspergillus flavus Biological control of the trichoderma pseudokiningii bacterium TC-72 described in a claim 1。
10. the application in Aspergillus flavus on field control Semen arachidis hypogaeae of the microbial organic fertilizer according to any one of a claim 2-8, it is characterised in that application process is: microbial organic fertilizer preparation is applied with fertilizer when the peanut seeding with every mu 40-60 kilogram。
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| CN113481104A (en) * | 2021-07-14 | 2021-10-08 | 暨南大学 | Aspergillus flavus-resistant crop growth-promoting spore-free trichoderma preparation and application thereof |
| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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| CN113481104A (en) * | 2021-07-14 | 2021-10-08 | 暨南大学 | Aspergillus flavus-resistant crop growth-promoting spore-free trichoderma preparation and application thereof |
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| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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