CN104788175A - Biological trichoderma viride granule as well as preparation method and application thereof - Google Patents
Biological trichoderma viride granule as well as preparation method and application thereof Download PDFInfo
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- 239000008187 granular material Substances 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 241000223261 Trichoderma viride Species 0.000 title abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 49
- 230000004151 fermentation Effects 0.000 claims abstract description 49
- 240000008042 Zea mays Species 0.000 claims abstract description 47
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 45
- 239000003337 fertilizer Substances 0.000 claims abstract description 31
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 16
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 16
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000004021 humic acid Substances 0.000 claims abstract description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000015099 wheat brans Nutrition 0.000 claims abstract description 11
- WZLMXYBCAZZIRQ-UHFFFAOYSA-N [N].[P].[K] Chemical compound [N].[P].[K] WZLMXYBCAZZIRQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 241000223259 Trichoderma Species 0.000 claims description 149
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 41
- 239000003795 chemical substances by application Substances 0.000 claims description 37
- 239000002245 particle Substances 0.000 claims description 31
- 239000000843 powder Substances 0.000 claims description 26
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 23
- 201000010099 disease Diseases 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 23
- 235000009973 maize Nutrition 0.000 claims description 23
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- 210000000582 semen Anatomy 0.000 claims description 20
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- 238000001035 drying Methods 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 241001460073 Trichoderma asperellum Species 0.000 claims description 16
- 239000002054 inoculum Substances 0.000 claims description 16
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 16
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- Fertilizers (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
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Abstract
The invention discloses a trichoderma viride granule as well as a preparation method and application thereof. The trichoderma viride granule comprises the following components in percentage by weight: 30-40 percent of trichoderma viride liquid fermentation liquid, 5-10 percent of corn flour, 30-40 percent of diatomite, 10-20 percent of wheat bran, 1-2.0 percent of zinc sulfate fertilizer, 0.4-0.6 percent of humic acid and 0.4-0.6 percent of nitrogen phosphorus potassium compound fertilizer. The biological granule has the advantages of high antagonism and high specificity, the com stalk rot can be effectively prevented by using the granule, and multiple coordination effects of restraining germination of pathogenic bacteria in soil, promoting breeding of trichoderma viride and promoting crop root growth are achieved.
Description
Technical field
The present invention relates to a kind of Biocontrol Effect microbial inoculum, be specially a kind of Trichoderma biological particles agent and its preparation method and application, the preparation method of particularly a kind of Trichoderma biological particles agent of control of maize stem rot and control and application thereof.
Background technology
Trichoderma (Trichoderma viride) a kind ofly applies quite general biological control microorganism in the world, as the Antagonism fungi that tool one class has important biological and ecological methods to prevent plant disease, pests, and erosion to be worth, just have been found that there is antagonistic action to plant pathogenic fungi as far back as the thirties in 20th century, its mechanism of action comprises generation antibiosis, hyperparasitism, bacteriolysis, Competition etc.
Corn is the first food crop of China, corn stalk rot disease is the soil-borne disease of a class serious threat Maize Production, causes serious lodging and production loss, at present for the method that the control of this disease mainly takes chemical pesticide to dress seed, but preventive effect generally lacks good, and preventive effect majority is only about 30%.On the other hand, along with China is to the increase of the demand of diseases and pests of agronomic crop green prevention and control, the life-time service of chemical pesticide has caused detrimentally affect in various degree to ecotope.The occurrence and harm how utilizing biological control microbiological manipulation maize diseases is the technological breakthrough realizing maize diseases green prevention and control, seeks a kind of biological control method that can reduce chemical pesticide use and obtains most attention.Trichoderma is a kind of resourceful biocontrol fungi be extensively present in soil, can not only be widely used in all kinds of soil-borne disease of crop of control, can also Promoting plant growth, increase yield, and reduce soil agrochemistry and pollute, application is had a bright future.Therefore, develop the biocontrol trichoderma microbial inoculum with good biological and ecological methods to prevent plant disease, pests, and erosion and production-increasing function soil-borne disease green prevention and control tool is of great significance.And these preparations are generalized to national each major grain producing area there is extensive prospect.
The Trichoderma microbial inoculum majority of current domestic registration is wettable powder, mainly be registered on vegetables and flowers plant and (as fertile trichoderma harzianum wettable powder is visitd by the U.S., register the root rot of crop greenhouse bird watching and damping-off, the samping off of tomato.The trichoderma wettable powder of Shangdong Tenov Pesticides Co., Ltd., the main eliminating vegetable botrytis that is registered in is prevented and treated), and this wettable powder is not suitable for process soil and corn seed dressing, on the one hand at present trichoderma wettable powder mainly with to the conidium of environment sensitive for main component, on the other hand, trichoderma wettable powder directly processes corn seed easily affects seed germination, and Trichoderma conidiocidal activity is subject to the impact (the corn seed great majority various chemical seed coat agent of dressing all that domestic all commercialization is sold) of the seed-coat chemical seed coat agent of dressing.The main resistance chlamydospore of effective constituent that Trichoderma Granular formulations is produced, be applicable to the various soil adverse circumstance factor, have obvious resistance, can survive 6 months under arid edaphic condition, and around the seed that can be applied to the chemistry of dressing seed coat agent, and its activity is unaffected.In addition, zinc fertilizer in Trichoderma granule and the component of nitrogen-phosphorus-potassium compound fertilizer except for Trichoderma soil decided at the higher level but not officially announced grow nutrition is provided except, also can be converted in soil by Trichoderma can by the biochemical substances of the quick-acting nutritious of corn efficiency utilization and inducing maize immunity, thus inducing maize is to the systemic immunity ability of stem rot.
Summary of the invention
The object of this invention is to provide a kind of Trichoderma biological particles agent and its preparation method and application, be specially a kind of preparation method and biocontrol effect of the Trichoderma biocontrol fungicide for control of maize soil-borne disease stem rot.Trichoderma preparation of the present invention can be effective to control of maize soil-borne disease stem rot.
The present invention passes through large quantity research for many years and finds Trichoderma asperellum ZJSX5003 (Chinese biological engineering magazine, 2013,33
(2): 97-104), competitive violently viability, to the hyperparasitism of pathogenic bacteria in soil and the ability of induction of resistance.
The object of the invention is to be achieved through the following technical solutions: the biocontrol microorganisms adopted is Trichoderma ZJSX5003, be transferred to after purified in test tube slant and make female microbial inoculum, in triangular flask, shaking culture is liquid spawn, carrying out solid fermentation with nutraceutical matrix Semen Maydis powder and inorganic salts again makes it produce trichoderma liquid fermented liquid, make Trichoderma biological prevention and control agent after being mixed with carrier by the fermented liquid being loaded with a large amount of Trichoderma spore, potted plant with field sowing before carry out the control that soil spreads manuer in holes for corn stalk rot disease there is notable synergistic effect.
First aspect, the invention provides the agent of a kind of Trichoderma biological particles, and the agent of described Trichoderma biological particles comprises following component and weight content:
Preferably, being prepared as follows of described trichoderma liquid fermented liquid: fermention medium is loaded in fermentor tank, at 121 DEG C, sterilizing 30 ~ 60 minutes, accesses the second class inoculum of 0.5% weight ratio, becomes green and trichoderma liquid fermented liquid in 28 DEG C of condition bottom fermentation to fermention mediums.
Preferably, described fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor;
The preparation of described fermention medium is specially: first potassium primary phosphate, magnesium sulfate, manganous sulfate, zinc sulfate, SODIUMNITRATE, ammonium sulfate, sodium-chlor are dissolved in water, then adds sterilizing 30 ~ 60 minutes and get final product at 121 DEG C after Semen Maydis powder mixing.
Preferably, the volume of described fermentor tank is 300L.
Preferably, the time of described fermentation is 7 days.
Preferably, being prepared as follows of described second class inoculum: access dull and stereotyped bacterial classification in sterilized second order fermentation substratum, be 3 days in 28 DEG C, 180 revs/min condition bottom fermentation times, obtaining second class inoculum, is the conidium liquid spawn of cotton-shaped mycelia and the dispersion of dispersing in a large number.
Preferably, described second order fermentation substratum is for getting potato 200 grams, glucose 20 grams, 1 liter, water, and adjust pH is 6 ~ 8; Described second order fermentation substratum to be divided in 250 milliliters of triangular flasks 100 milliliters; Described sterilizing to refer at 121 DEG C sterilizing 30 minutes.
Preferably, being prepared as follows of described dull and stereotyped bacterial classification: be inoculated in by Trichoderma asperellum ZJSX5003 in PDA substratum plate, in 28 DEG C of incubators, is inverted cultivation 2 ~ 3 days, obtains final product.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, retain juice; Add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
Be organic whole due to component each in Trichoderma granule and have obvious synergistic function.The organic acid that Trichoderma produces, polysaccharide, protein and phytase improve content and the plant absorption ability of soluble phosphorus & potassium in soil, and potassium is mainly plant epidermis cell walls and provides xylogen, improves the resistivity to pathogen infection.Semen Maydis powder is mainly Trichoderma determining in soil and on root system of plant and grows raising carbon source.Humic acid, can also stimulating plant growth and disease resistance except the soluble potassium in soil in can increasing and phosphorus.Except above-mentioned functions, the maximum feature of the technical program creates new function after the combination of each composition: (1) makes solubility nutrition in soil increase due to Trichoderma granule, accelerate Trichoderma and surely grow efficiency at Rhizosphere of Crops soil and root table, it grows efficiency (5 leaf phase seedling age) surely from 40 ~ 50% of single trichoderma component (wooden mould fermented liquid+diatomite), bring up to 80 ~ 90% (entirely combining), enable Trichoderma from seedling stage-Adult plant (filling stage) whole-process high-efficiency grows surely, capture ecological niche, stop infecting of germ; (2) research finds that humic acid component can stimulate Trichoderma to produce conidium; (3) humic acid improves Trichoderma component induction root system generation xylogen ability more than two times; (4) research finds that the wheat bran of granule and Semen Maydis powder obviously induce Trichoderma cellulase activity to improve, and improve 30%, and we proves that cellulase is the exciton of system induction corn resistance first than single Trichoderma component.
Second aspect, the invention provides the preparation method of a kind of described Trichoderma biological particles agent, and described preparation method is as follows:
Raw material trichoderma liquid fermented liquid 30 ~ 40%, Semen Maydis powder 5 ~ 10%, diatomite 30 ~ 40%, wheat bran 10 ~ 20%, zinc sulfate fertilizer 1 ~ 2.0%, humic acid 0.4 ~ 0.6%, nitrogen-phosphorus-potassium compound fertilizer 0.4 ~ 0.6% are stirred, then extruder grain, under 43 ~ 50 DEG C of conditions, namely dry 1 ~ 2h obtains Trichoderma granule.
Preferably, in described stirring, every batch of weight stirring raw material is: 9 ~ 10kg trichoderma liquid fermented liquid and 10 ~ 12kg solid materials; Described solid materials is Semen Maydis powder, diatomite, wheat bran, zinc sulfate fertilizer, humic acid, nitrogen-phosphorus-potassium compound fertilizer.
Preferably, the rotating speed of described stirring is 40 ~ 50 turns/min.
Preferably, described stirring specifically adopts vertical dough mixing machine, model HLN25.
Preferably, in described granulation, every batch of granulation weight is 25kg, and prilling temperature is 25 ~ 30 DEG C, and particle water content is 30 ~ 40wt%.
Preferably, described granulation specifically adopts Squeezinggranulator, model XL-300.
Preferably, in described drying, every batch of dry weight is 50kg, and drying temperature is 45 ~ 50 DEG C, and time of drying is 40 ~ 60min, and after dry, the water content of gained Trichoderma biological particles agent is 10 ~ 20wt%.
Preferably, described drying specifically adopts ebullated dryer, model: FG-120.
The quality standard of the Trichoderma biological particles agent that this preparation method prepares is specially: particle diameter 0.15 ~ 0.2cm, and smooth surface is hard, water content 10 ~ 20%, pH5 ~ 7, spore amount 2 ~ 4 × 10 of living
8cFU/g, particle 6 ~ 10 minutes can be entirely soluble in water, is suitable for mechanize and mixes with chemical fertilizer and use.
The third aspect, the invention provides the application of a kind of described Trichoderma biological particles agent in control of maize soil-borne disease stem rot.
Preferably, described application specifically refers to: field test to be spreaded manuer in holes the agent of described Trichoderma biological particles with the consumption on 5 kilograms, every mu of ground; Pot experiment application process is basin (diameter 20cm) every before sowing, Trichoderma biological particles agent 6 grams of spreading manuer in holes.
Preferably, spread manuer in holes described in as described Trichoderma biological particles agent of spreading manuer in holes before sowing.
Compared with prior art, the present invention has following beneficial effect:
1. trichoderma strain is mixed and made into biological and ecological methods to prevent plant disease, pests, and erosion granule through fermentation and carrier, and making method is simple, is suitable for different scales and produces;
2. Trichoderma granule is the soil improvement agent of the continuous cropping obstacle that control of maize soil-borne disease stem rot causes, Trichoderma is made biological prevention and control agent and is manured into soil, both be conducive to improving edatope, be conducive to again the stretching, extension of mycelia in soil to spread, in the prolific process of Trichoderma, suppress the growth of caused by soil germ, solve soil-borne disease and Soil-sickness Problem simultaneously;
3. according to granule provided by the invention, be manured into soil crop rhizosperic prior to seeding, and multiply further at soil along with the growth of root system, noresidue, pollution-free, in Haicheng City testing station, testing station of Jiangsu Dafeng City, Zhaodong, Heilungkiang testing station, Chengdu, Sichuan testing station, Meihekou testing station, Tonghua, zunyi, guizhou testing station, Yinchuan of Ningxia Province testing station field test, the stem rot sickness rate of granule treatment group can reach more than 55% than the low preventive effect of control group;
4. the present invention is applicable to most of trichoderma strain, and making method is simple, water and electric saving, is suitable for different scales and produces, wide market;
5. be easy to transport preservation, for shelf-stable, can long-acting in corn stalk rot disease preventive and therapeutic effect.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 (a) (b) Trichoderma asperellum ZJSX5003 live body of the present invention seedling suppresses Corn stalk rot pathogens and promotes the result schematic diagram of growth of maize; C () is the result schematic diagram of Trichoderma asperellum ZJSX5003 live body strain suppression Corn stalk rot pathogens; Wherein left, center, right are respectively the application Trichoderma granule, company limited of large well biological section Trichoderma fertilizer, blank process;
Fig. 2 is the result schematic diagram that Trichoderma asperellum ZJSX5003 field of the present invention suppresses stem rot biocontrol effect;
Fig. 3 is that the single trichoderma of Trichoderma asperellum ZJSX5003 of the present invention is with combining trichoderma grows comparing of efficiency surely at Rhizosphere of Crops soil and root table entirely;
Fig. 4 is that Trichoderma asperellum ZJSX5003 mixture pellets of the present invention compares with single particle agent cellulase activity.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment provides the preparation method of a kind of Trichoderma biological particles agent, specifically comprises the steps:
1, dull and stereotyped bacterial classification: Trichoderma asperellum ZJSX5003 is inoculated in PDA substratum plate, in 28 DEG C of incubators; Be inverted cultivation 2 ~ 3 days.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, retain juice; Add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
2, the preparation of second class inoculum: get potato 200 grams, glucose 20 grams, 1 liter, water, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 3 days, the cotton-shaped mycelia of being dispersed in a large number and the conidium liquid spawn of dispersion.
3, prepared by trichoderma liquid fermented liquid: fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor, first inorganic salt are dissolved in water, solid fermentation substratum is made after fully being mixed with water by culture material again, solid fermentation substratum loads in 300 liters of fermentor tanks, sterilizing 30 ~ 60 minutes at 121 DEG C, the second class inoculum of access part by weight 0.5%, controlling leavening temperature is 28 DEG C, fermentation time 7 days, green and trichoderma liquid fermented liquid is become to culture material,
4, Trichoderma granule preparation: trichoderma liquid fermented liquid 40%, Semen Maydis powder 5.2%, diatomite 40%, wheat bran 13%, zinc sulfate fertilizer 1%, humic acid 0.4%, nitrogen-phosphorus-potassium compound fertilizer 0.4% use stirrer to stir; after tablets press extruder grain, drying machine 43 ~ 50 DEG C of dryings 1 ~ 2 hour finished product is Trichoderma granule.
5, live body Maize at Seedling Stage experiment: every basin weighs same matrix, plantation Zheng Dan 958 corn seed, before treatment group plantation, every basin soil is spreaded manuer in holes the granule of 6g different ingredients, so that the biological board Trichoderma of large well is fertile in contrast at present.Normal plantation as blank, the strain of every basin 5, often process 10 basins, potted plantly emerge after 50% until all, every 7 days record seedling rates, corn records Maize at Seedling Stage plant height tri-leaf period, all potted plant corn grain of hindering the inoculation of root method landfill and containing stem rot germ, record sickness rate after 7d, disease index, calculate preventive effect, measure fresh weight, dry weight, root depth.
6, Adult plant inoculation test: every basin weighs same matrix, before treatment group plantation, every basin soil is spreaded manuer in holes 6 grams of Trichoderma asperellum ZJSX5003 granules, with conventional Trichoderma fertilizer (great Jing bio tech ltd) and space management for contrast, the strain of every basin 5, often process 5 basins, typhon mouth phase potted plant basal part of stem is inoculating two kinds pathogenic bacteria germ (hindering the corn grain of root method landfill inoculation containing stem rot germ) respectively, adds up biocontrol effect after morbidity.
Table 1
Trichoderma asperellum ZJSX5003 investigates the biocontrol effect of corn stalk rot disease:
Diseased plant rate=diseased plant rate/investigate total strain number × 100%
Biocontrol effect=[(diseased plant rate of contrast diseased plant rate-process)/contrast diseased plant rate] × 100%
In the experiment of live body Maize at Seedling Stage: as Fig. 1 (a) is depicted as blank group and Trichoderma granule treatment difference, Trichoderma granule treatment is thicker, many than blank group root system, plant is high, blade is large (left side is blank, and right side is Trichoderma granule treatment of the present invention).Difference is processed as Fig. 1 (b) is depicted as company limited of blank group and large well biological section Trichoderma fertilizer, the Trichoderma fertilizer process of company limited of large well biological section is thicker, many than blank group root system, but fewer than Trichoderma granule treatment (left side is blank, and right side is the Trichoderma fertilizer process of company limited of large well biological section).The seedling rate of Trichoderma granule treatment, plant height, fresh weight, dry weight, root are long is respectively 96%, 14.50 centimetres, 1.649 grams, 0.208 gram, 10.82 centimetres; Company limited of large well biological section Trichoderma fertilizer is respectively 94%, 13.1 centimetres, 1.5 grams, 0.1 gram, 9.0 centimetres.Blank group is respectively 94%, 12.74 centimetres, 1.386 grams, 0.193 gram, 10.24 centimetres.Sickness rate, the disease index of Trichoderma granule treatment group are respectively 14%, 21; Company limited of large well biological section Trichoderma fertilizer is respectively 30%, 40; Blank group is respectively 62%, 77.Trichoderma granule and company limited of large well biological section Trichoderma fertilizer are respectively 72.73%, 48% to the anti-of stem rot.Trichoderma granule treatment is all better than the contrast of conventional bacterial manure and blank in seedling growth of corn index and preventive effect.
Adult plant inoculation test: as shown in Fig. 1 (c), wherein left, center, right are respectively the application Trichoderma granule, company limited of large well biological section Trichoderma fertilizer, blank process; The sickness rate of Trichoderma granule treatment, disease index are respectively 40%, 21, and the sickness rate of company limited of large well biological section Trichoderma fertilizer and disease index, be respectively 65% and 40, blank group is respectively 80%, 60.Trichoderma granule and company limited of large well biological section Trichoderma fertilizer are respectively 65% and 33% to the anti-of stem rot.Trichoderma granule treatment is all better than contrast to seedling growth of corn and stem rot preventive effect.
embodiment 2
The present embodiment provides the preparation method of a kind of Trichoderma biological particles agent, specifically comprises the steps:
1, dull and stereotyped bacterial classification: be inoculated in by Trichoderma asperellum ZJSX5003 in PDA substratum plate, in 28 DEG C of incubators, is inverted cultivation 2 ~ 3 days.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, and retain juice, add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
2, the preparation of second class inoculum: get potato 200 grams, glucose 20 grams, 1 liter, water, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 3 days, the cotton-shaped mycelia of being dispersed in a large number and the conidium liquid spawn of dispersion.
3, prepared by trichoderma liquid fermented liquid: fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor, first inorganic salt are dissolved in water, solid fermentation substratum is made after fully being mixed with water by culture material again, solid fermentation substratum loads in 300 liters of fermentor tanks, sterilizing 30 ~ 60 minutes at 121 DEG C, the second class inoculum of access part by weight 0.5%, controlling leavening temperature is 28 DEG C, fermentation time 7 days, green and trichoderma liquid fermented liquid is become to culture material,
4, Trichoderma granule preparation: trichoderma liquid fermented liquid 30%, Semen Maydis powder 10.0%, diatomite 36.8%, wheat bran 20%, zinc sulfate fertilizer 2.0%, humic acid 0.6%, nitrogen-phosphorus-potassium compound fertilizer 0.6%; use stirrer stirs; after tablets press extruder grain, drying machine 43 ~ 50 DEG C of dryings 1 ~ 2 hour finished product is Trichoderma granule.
5, field test: process designs with model field: select area in flakes, smooth, the uniform field of fertility of physical features does model field.Establish three process altogether: Trichoderma granule treatment, blank, local chemical seed coat agent.Field management is identical with local production field.
Granule treatment method: kind is local main breed, in Haicheng City testing station, beautiful No. 8 first beautiful 335 of single 76, No. 9, Zhong Yu No. 3, day agriculture of testing station of Jiangsu Dafeng City, Zhaodong, Heilungkiang testing station, Chengdu, Sichuan testing station, Meihekou testing station, Tonghua, zunyi, guizhou testing station, Yinchuan of Ningxia Province testing station auspicious moral 999, Su Yu 29, the dragon planted respectively, benefit.Be manured into soil in granule cave respectively in sowing time, then sow, use 5kg for every mu, planting density is 4500 strain every mu, and applying quantity of chemical fertilizer is by local actual application level.
Field efficacy is investigated: at Adult plant (after milk stage), often repeat to get 3 ridges at random, 20 meters of ridges are long, random searching 20 strain of every ridge, and three are repeated 60 strains altogether, investigates the diseased plant rate of each repetition, statistics field efficacy.
Diseased plant rate %=diseased plant rate/investigate total strain number × 100%
Preventive effect=[(diseased plant rate of contrast diseased plant rate-process)/contrast diseased plant rate] × 100%
Result as shown in Figure 2, in Haicheng City testing station, testing station of Jiangsu Dafeng City, Zhaodong, Heilungkiang testing station, Chengdu, Sichuan testing station, Meihekou testing station, Tonghua, zunyi, guizhou testing station, Yinchuan of Ningxia Province testing station granule treatment preventive effect be respectively 66.67%, 63.64%, 66.67%, 70.46%, 55.5%, 55.67%, 100%, and seed coat agent flue god preventive effect is generally 35 ~ 40%.
embodiment 3
1, dull and stereotyped bacterial classification: Trichoderma asperellum ZJSX5003 is inoculated in PDA substratum plate, in 28 DEG C of incubators; Be inverted cultivation 2 ~ 3 days.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, retain juice; Add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
2, the preparation of second class inoculum: get potato 200 grams, glucose 20 grams, 1 liter, water, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 3 days, the cotton-shaped mycelia of being dispersed in a large number and the conidium liquid spawn of dispersion.
3, prepared by trichoderma liquid fermented liquid: fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor, first inorganic salt are dissolved in water, solid fermentation substratum is made after fully being mixed with water by culture material again, solid fermentation substratum loads in 300 liters of fermentor tanks, sterilizing 30 ~ 60 minutes at 121 DEG C, the second class inoculum of access part by weight 0.5%, controlling leavening temperature is 28 DEG C, fermentation time 7 days, green and trichoderma liquid fermented liquid is become to culture material,
4, Trichoderma granule preparation: single trichoderma component: trichoderma liquid fermented liquid 37.8%, diatomite 62.2%.Full combination trichoderma component: trichoderma liquid fermented liquid 37.8%, Semen Maydis powder 9%, diatomite 40%, wheat bran 10%, zinc sulfate fertilizer 2%, humic acid 0.6%, nitrogen-phosphorus-potassium compound fertilizer 0.6%; Use stirrer stirs, and after tablets press extruder grain, drying machine 43 ~ 50 DEG C of dryings 1 ~ 2 hour finished product is Trichoderma granule.
5, live body Maize at Seedling Stage experiment: every basin weighs same matrix, plantation Zheng Dan 958 corn seed, before treatment group plantation, every basin soil is spreaded manuer in holes the granule of 6g different ingredients, with current single trichoderma for contrast.The strain of every basin 5, often processes 10 basins, and the corn five leaf phase measures Maize at Seedling Stage Trichoderma and surely grows efficiency at rhizosphere soil and root table
6, measure Maize at Seedling Stage Trichoderma and surely grow efficiency: Trichoderma grows efficiency surely at rhizosphere soil: often process and get Zea mays root 3 to be measured strain respectively, gets 10 grams and adds in the triangular flask filling 90 ml sterile waters; The Rhizosphere Soil shaking off surface soil is got 5g, adds in the triangular flask filling 45 ml sterile waters, 150r/min, vibration 30min, making native content is 10
-1g/mL Soil Slurry; Draw 1 milliliter before not precipitating to add another and fill in the test tube of 9 ml sterile waters and shake up, and rare to 10 successively
-5g/mL, respectively absorption 0.2 milliliter add on rose-bengal streptomycin medium flat board good in advance, smear evenly with sterilizing glass stick.Often process repetition 3 ware.Record the colony count on substratum after cultivating 24h, and analyze the population density of soil microorganisms according to colony count.
Trichoderma grows efficiency surely at root table: repeatedly rinse with sterilized water after maize root system tap water is clean, thorough removal Rhizosphere Soil, the maize root system of different treatment is subtracted into the segment of about 0.5mm, homogenate is ground in mortar, be applied to the rose-bengal streptomycin medium prepared after dilution, after cultivating 24h, record the colony count on substratum.
The results are shown in Figure 3, because Trichoderma granule makes solubility nutrition in soil increase, accelerate Trichoderma and surely grow efficiency at Rhizosphere of Crops soil and root table, it grows efficiency (5 leaf phase seedling age) surely from 40 ~ 50% of single trichoderma component, bring up to 80 ~ 90% of full combination, Trichoderma was grown to Adult plant whole-process high-efficiency surely from seedling stage, captures ecological niche, stop infecting of germ.
embodiment 4
1, dull and stereotyped bacterial classification: Trichoderma asperellum ZJSX5003 is inoculated in PDA substratum plate, in 28 DEG C of incubators; Be inverted cultivation 2 ~ 3 days.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, retain juice; Add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
2, the preparation of second class inoculum: get potato 200 grams, glucose 20 grams, 1 liter, water, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 3 days, the cotton-shaped mycelia of being dispersed in a large number and the conidium liquid spawn of dispersion.
3, prepared by trichoderma liquid fermented liquid: fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor, first inorganic salt are dissolved in water, solid fermentation substratum is made after fully being mixed with water by culture material again, solid fermentation substratum loads in 300 liters of fermentor tanks, sterilizing 30 ~ 60 minutes at 121 DEG C, the second class inoculum of access part by weight 0.5%, controlling leavening temperature is 28 DEG C, fermentation time 7 days, green and trichoderma liquid fermented liquid is become to culture material,
4, single trichoderma component: wooden mould liquid fermentation liquid 40%, diatomite 60%; Compound trichoderma component: trichoderma liquid fermented liquid 40%, Semen Maydis powder 20.0%, diatomite 30%, wheat bran 10% use stirrer to stir, after tablets press extruder grain, drying machine 43 ~ 50 DEG C of dryings 1 ~ 2 hour finished product is Trichoderma granule.
5, live body Maize at Seedling Stage experiment: every basin weighs same matrix, plantation Zheng Dan 958 corn seed, before treatment group plantation, every basin soil is spreaded manuer in holes the granule of 6g different ingredients, with single trichoderma for contrast.Normal plantation as blank, the strain of every basin 5, often processes 10 basins, and often process is got root samples and carried out cellulase activity vitality test
6, cellulase activity measures
Dull and stereotyped bacterial classification: repeatedly rinse with sterilized water after sample root system tap water is clean, the maize root system of different treatment is subtracted into the segment of about 0.5mm, be ground into homogenate in mortar, be applied to the rose-bengal streptomycin medium prepared after dilution,
(1) potato 200 grams, glucose 20 grams, 1 liter, water is got, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 2d, grows prosperity to mycelium.Vacuum filtration is except supernatant liquor, and namely precipitation plants daughter bacteria.
(2) take some parts of 1g kind daughter bacterias, be transferred to and produce in enzyme induction substratum.28 DEG C, 180rpm constant-temperature shaking culture 4d.Sample 1 milliliter every day, centrifugal 10 minutes of 6000rpm.Get 50 μ L zytase fermented liquids (zytase inducing culture supernatant liquor is contrast), Acetic acid-sodium acetate damping fluid (0.1mol/L, pH 5.0) is diluted to 200 μ L, adds 1.8 milliliter of 1% xylan solution substrate, abundant mixing, 50 DEG C of water-baths 10 minutes.Add 3 milliliters of DNS agent, boiling water bath reacts 10 minutes, measures OD after dilution
540nmlight absorption value.Test 3 repetitions.For Trichoderma asperellum Xylanase activity detected result, reach maximum value at fermentation 3d.
(3) xylose standard curve preparation with reference to (Fei Dibo, Feng Guanquan, Yuan Chao. the research of Xylanases in Feedstuffs activity determination method. Zhejiang Agriculture journal, 2004,1:53-58).Enzyme activity unit defines: under these conditions, per minute produces enzyme amount needed for 1 μm of ol wood sugar and is decided to be 1 enzyme activity unit.
Acetic acid-sodium acetate damping fluid (0.1mol/L, pH 5.0): draw 1.8 milliliters, Glacial acetic acid, add suitable quantity of water, add sodium-acetate 5.78 grams, dissolve, ultrapure water is settled to 1000 milliliters, uses after regulating PH to 5.0.
1% xylan substrate: take xylan 1.00 grams in beaker, pasty state is soaked into 2 milliliters of 0.5mol/L sodium hydroxide, add 40 milliliters of Acetic acid-sodium acetate damping fluid (0.1mol/L, pH 5.0), heating for dissolving in 60-70 DEG C of water-bath, with 0.5mol/L acetic acid (about 2mL-sodium-acetate buffer (0.1mol/L after cooling, p-sodium-acetate buffer (0.1mol/L, pH 5.0) be settled to 100 milliliters, stir, be stored in-20 DEG C of refrigerators for subsequent use.
As shown in Figure 4, mixture pellets comparatively improves than single particle agent cellulase activity, and improve 30% than single Trichoderma component, in mixture pellets, component wheat bran and Semen Maydis powder obviously induce Trichoderma cellulase activity to improve.
embodiment 5
1, dull and stereotyped bacterial classification: Trichoderma asperellum ZJSX5003 is inoculated in PDA substratum plate, in 28 DEG C of incubators; Be inverted cultivation 2 ~ 3 days.
PDA substratum: 200 grams of potatoes are cut into 1cm
3fritter, little fire boils 30 minutes, 4 layers of filtered through gauze, retain juice; Add 20 grams of glucose, 20 grams of agar powders, distilled water is settled to 1 liter, 121 DEG C of high pressure steam sterilizations 30 minutes.
2, the preparation of second class inoculum: get potato 200 grams, glucose 20 grams, 1 liter, water, be mixed with second order fermentation nutrient solution, adjust pH is 6 ~ 8, to be divided in 250 milliliters of triangular flasks 100 milliliters, at 121 DEG C, sterilizing 30 minutes, accesses dull and stereotyped bacterial classification, shaking speed is 180 revs/min, leavening temperature 28 DEG C, fermentation time is 3 days, the cotton-shaped mycelia of being dispersed in a large number and the conidium liquid spawn of dispersion.
3, prepared by trichoderma liquid fermented liquid: fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor, first inorganic salt are dissolved in water, solid fermentation substratum is made after fully being mixed with water by culture material again, solid fermentation substratum loads in 300 liters of fermentor tanks, sterilizing 30 ~ 60 minutes at 121 DEG C, the second class inoculum of access part by weight 0.5%, controlling leavening temperature is 28 DEG C, fermentation time 7 days, green and trichoderma liquid fermented liquid is become to culture material,
4, Trichoderma granule preparation:
Former trichoderma component: trichoderma liquid fermented liquid 30%, Semen Maydis powder 8%, diatomite 40%, wheat bran 20%, zinc sulfate fertilizer 1%, nitrogen-phosphorus-potassium compound fertilizer 0.6%.
Improvement trichoderma component: add humic acid 0.4% to described former trichoderma component.Use stirrer stirs, and after tablets press extruder grain, drying machine 43 ~ 50 DEG C of dryings 1 ~ 2 hour finished product is Trichoderma granule.Different treatment granule is got 1 gram, adds in the triangular flask filling 99 ml sterile waters, 150r/min, vibrate 30 minutes, making native content is 10
-2g/mL Soil Slurry; Do not precipitate front absorption, count with blood counting chamber.
5, live body Maize at Seedling Stage experiment: every basin weighs same matrix, plantation Zheng Dan 958 corn seed, before treatment group plantation, every basin soil is spreaded manuer in holes the granule of 6 grams of different ingredients, with current former trichoderma for contrast.Normal plantation as blank, the strain of every basin 5, often processes 10 basins, often process get 0.5 gram, the fresh sample of root, grind to form homogenate with the ethanol of 95%, 4500rpm is collecting precipitation after centrifugal 10 minutes.Precipitate 3 times with 95% alcohol flushing, then use volume ratio ethanol: normal hexane=1:1 rinses 3 times, collecting precipitation is also dry.Dry thing to be jumped a queue insulation 30 minutes with 0.5 milliliter of 25% acetobrom (glacial acetic acid configuration) dissolving, 70 DEG C of water-baths, then add 0.9 milliliter of 2mol/L NaOH termination reaction and add 5 milliliters of glacial acetic acids and 0.1 milliliter of 7.5mol/L oxammonium hydrochloride, after mixing, 4500rpm centrifugal 5 minutes Aspirate supernatant 0.1 milliliter add 3.0 milliliters of glacial acetic acid dilution metering OD
280nmlight absorption value represents the content of xylogen with A280nmmg protein-1.
Former trichoderma, improvement trichoderma are measured conidium number and are respectively 2.3 × 10
8cFU/g, 5.8 × 10
8cFU/g, finds that humic acid component can stimulate Trichoderma to produce more conidiums.It is more than the twice of former trichoderma that content of lignin improves trichoderma in measuring, and humic acid improves Trichoderma component induction root system and produces xylogen ability more than two times.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. the agent of Trichoderma biological particles, is characterized in that, the agent of described Trichoderma biological particles comprises following component and weight content:
2. Trichoderma biological particles according to claim 1 agent, it is characterized in that, being prepared as follows of described trichoderma liquid fermented liquid: fermention medium is loaded in fermentor tank, sterilizing 30 ~ 60 minutes at 121 DEG C, access the second class inoculum of 0.5% weight ratio, become green and trichoderma liquid fermented liquid in 28 DEG C of condition bottom fermentation to fermention mediums.
3. Trichoderma biological particles according to claim 2 agent, it is characterized in that, described fermentation medium components is: Semen Maydis powder 10 kilograms, 210 kilograms, water, potassium primary phosphate 766 grams, 100 grams, magnesium sulfate, manganous sulfate 0.5 gram, 0.4 gram, zinc sulfate, SODIUMNITRATE 284 grams, 220 grams, ammonium sulfate, 200 grams, sodium-chlor.
4. Trichoderma biological particles according to claim 2 agent, it is characterized in that, being prepared as follows of described second class inoculum: access dull and stereotyped bacterial classification in sterilized second order fermentation substratum, is 3 days in 28 DEG C, 180 revs/min condition bottom fermentation times, obtains second class inoculum.
5. Trichoderma biological particles according to claim 4 agent, is characterized in that, described second order fermentation substratum is for getting potato 200 grams, glucose 20 grams, 1 liter, water, and adjust pH is 6 ~ 8;
Being prepared as follows of described dull and stereotyped bacterial classification: be inoculated in by Trichoderma asperellum ZJSX5003 in PDA substratum plate, in 28 DEG C of incubators, is inverted cultivation 2 ~ 3 days, obtains final product.
6. a preparation method for Trichoderma biological particles according to claim 1 agent, is characterized in that, described preparation method is as follows:
Raw material trichoderma liquid fermented liquid 30 ~ 40%, Semen Maydis powder 5 ~ 10%, diatomite 30 ~ 40%, wheat bran 10 ~ 20%, zinc sulfate fertilizer 1 ~ 2.0%, humic acid 0.4 ~ 0.6%, nitrogen-phosphorus-potassium compound fertilizer 0.4 ~ 0.6% are stirred, then extruder grain, under 43 ~ 50 DEG C of conditions, namely dry 1 ~ 2h obtains Trichoderma granule.
7. the preparation method of Trichoderma biological particles according to claim 6 agent, is characterized in that, in described stirring, every batch of weight stirring raw material is: 9 ~ 10kg trichoderma liquid fermented liquid and 10 ~ 12kg solid materials;
The rotating speed of described stirring is 40 ~ 50 turns/min.
8. the preparation method of Trichoderma biological particles according to claim 6 agent, is characterized in that, in described granulation, every batch of granulation weight is 25kg, and prilling temperature is 25 ~ 30 DEG C.
9. the preparation method of Trichoderma biological particles according to claim 6 agent, is characterized in that, in described drying, every batch of dry weight is 50kg, and drying temperature is 45 ~ 50 DEG C, and time of drying is 40 ~ 60min.
10. the application of a Trichoderma biological particles according to claim 1 agent in control of maize soil-borne disease stem rot.
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| CN110142285B (en) * | 2019-05-24 | 2021-02-12 | 上海交通大学 | Method for remedying heavy metal contaminated soil by combination of trichoderma asperellum and alfalfa |
| CN115152773A (en) * | 2022-06-17 | 2022-10-11 | 安徽久易农业股份有限公司 | Composition of prothioconazole and fludioxonil for preventing and treating basal stem rot of wheat |
| CN115152773B (en) * | 2022-06-17 | 2023-12-29 | 安徽久易农业股份有限公司 | Composition of prothioconazole and fludioxonil for preventing and treating wheat stem rot |
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