CN103484384B - Trichoderma atroviride preparation for preventing and controlling vegetable fungal diseases and preparation method of trichoderma atroviride preparation thereof - Google Patents
Trichoderma atroviride preparation for preventing and controlling vegetable fungal diseases and preparation method of trichoderma atroviride preparation thereof Download PDFInfo
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Abstract
The invention relates to a trichoderma atroviride preparation for preventing and controlling vegetable fungal diseases and a preparation method thereofof the trichoderma atroviride preparation. A trichoderma strain Tr1208 is trichoderma atroviride and preserved in the CGMCC (China General Microbiological Culture Collection Center), and the preservation number is CGMCC No. 6828. According to The the trichoderma atroviride preparation takes, the trichoderma atroviride Tr1208 is used as an effective active ingredient and is prepared by being matched with an agriculturally acceptable carrier and a protective agent for the preparation; the bacteria content of the effective active ingredient is (1.0-30.0)*108 spores/gram; and the bacteria protectant content is 0.05%-1.8%. The preparation can have the effects of effectively preventing and controlling leek Botrytis, cucumber brown patches, cucumber powdery mildew, cucumber downy mildew, tomato early blight, cucumber type damping off, cucumber type blight and cucumber type root rot and promoting plant growth and characteristics of efficiency, wide antimicrobial spectrum, simple usage, low cost, no environmental pollution and plant micro ecological environment improvement.
Description
Technical field
The invention belongs to microbial pesticide technical field, be specifically related to Trichoderma atroviride preparation of a kind of antagonism vegetable pathogenic fungi and preparation method thereof and application.
Background technology
Along with the quickening of China Agricultural Structure Adjustment paces and the raising of living standards of the people, the variation of vegetable variety and the demand of anniversary stable market supply strengthen day by day, the scale of China's Vegetable produce constantly expands, output grows steadily, and has become in China's plant husbandry the second largest farm crop being only second to grain.According to Ministry of Agriculture's agriculture feelings statistics, within 2009, China's growing vegetables area accounts for 43% of the whole world, and ultimate production accounts for 49.6% of the world.Ultimate production and export volume all rank first in the world, and can be rated as the first in the world vegetables big country.
But along with the development of China's Vegetable produce, the diversity of cultivation mode, vegetable growing kind and kind rich, especially the variation of planting environment, cause some Minor diseases to rise, pathogenic bacteria host range expands, and creates many new expression urgently studied, some the old and new's diseases alternately cause harm, and these all seriously govern the development of China's vegetables industry.The wherein fungal disease such as leek gray mold, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class damping-off, melon and fruit class blight, melon and fruit class root rot, all occur comparatively serious, cause Severe Reduction, even have no harvest, become the restrictive barriers of Vegetable produce.Such disease of current control still mainly adopts chemical agent, and usually fresh out of medicine is just gathered sale, not only causes Vegetable pollution, also very easily causes resistance problems simultaneously.Therefore research and develop safety and environment microbial pesticide harmonious mutually and become inevitable, wherein wooden mould this kind of beneficial microorganism, because it has the resistance against diseases of wide spectrum and microecosystem regulation, induction of resistance effect, and studied for biocontrol of plant disease with improve plant growth.
Wood is mould is the ubiquitous class fungi of occurring in nature, and at soil, plant rhizosphere, leaf encloses and seed, bulb surface often can be separated to.It is found that in recent years some wood mould be bacterial parasite or the Antagonistic Fungi of plant pathogenic fungi, as trichoderma harziarum (
t. harzianum), hook-shaped wood mould (
t.hamatum), viride (
t. viride) can fungal disease be prevented and treated.Up to now about the mould research in biological and ecological methods to prevent plant disease, pests, and erosion of wood has carried out nearly 70 years, there is commercial Trichoderma preparation abroad.As with prevent and treat fruit trees in plum line silver leaf (
chondrostereum purpureum) Trichoderma preparation in West Europe merchandized handling; The biological prevention and control agent being fermented liquid with trichoderma harziarum T39 bacterial strain of the Makhteshim Agan company exploitation of Israel, trade(brand)name Trichodex, formulation is 25% can wet-milling, it may be used for control ash arrhizus bacteria (
botrytis cinerea), hyphal cluster germ (
scleratina sclerotiorum), leaf mold (
cladosporium fulvum) etc. disease, also have at home research and utilization Trichoderma control Plant diseases.21 century is called as environmental protection century, and the application of chemical agent is progressively restricted, and is therefore called hot issue to the biological control research nature of vegetable disease.
The present invention is just based on above reason, cause harm with current vegetables comparatively serious, lack again biological control means leek gray mold, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class damping-off, melon and fruit class blight, melon and fruit class root rot be target, from Microbial resources, find the microorganism with efficient antagonistic activity, be intended to be research and development new bio microbial inoculum and find new resource.
Summary of the invention
The present invention aims to provide a kind of Trichoderma atroviride preparation preventing and treating vegetable disease and preparation method thereof, may be used for preventing and treating vegetable fungi disease, solves pesticide residue, resistance and problem of environmental pollution that chemical pesticide control brings.The present invention can be used for control leek gray mold, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class damping-off, melon and fruit class blight, melon and fruit class root rot etc., has efficient, spectrum, stable performance, the vegetables microbiobacterial agent that is produced on a large scale.For realizing this object, the invention discloses following technology contents:
One strain has the Trichoderma atroviride strain of antagonism vegetable pathogenic fungi activity, it is characterized in that this bacterial strain is Trichoderma atroviride Tr1208(Trichoderma atroviride), in preservation China Committee for Culture Collection of Microorganisms common micro-organisms on November 15 center in 2012, preserving number has been CGMCCNo.6828.Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.
The Trichoderma atroviride strain Tr1208(of preservation of the present invention
trichoderma atroviride) physiology, biochemical characteristic as follows:
Trichoderma strain Tr1208 is rapid at PDA grow on plates, the flat board of bacterium colony covering diameter 90mm in 3 days, and radially, surface porosity is smooth; Within 3rd day, obviously have spore to produce as seen what cultivate, be initially light green, increase gradually later, present deep green, gonimoblast bundle is arranged in concentric wheel line.The mycelium that microscopic examination is cultivated is colourless, branch is complicated, is made up of the branch mycelia with tabula; The conidium produced is without barrier film, spherical in shape, and be polymerized to head arrangement, Dan Sheng, cell walls exists obvious and small coarse projection.Remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number is 1:2 ~ 5.
The present invention further discloses a kind of Trichoderma atroviride microbial inoculum, it is characterized in that it is is the Trichoderma atroviride Tr1208(of CGMCCNo.6828 by preserving number
trichoderma atroviride) thalline and its secondary metabolite be effective active composition, is equipped with agriculturally acceptable carrier and bacteria protectant composition; Wherein effective active composition is 1.0 ~ 30.0 × 10
8individual spore/gram, bacteria protectant content is 0.05 ~ 1.8%(w/w).
The preferred Trichoderma atroviride microbial inoculum of the present invention, it is characterized in that it is is the Trichoderma atroviride Tr1208(of CGMCCNo.6828 by preserving number
trichoderma atroviride) thalline is effective active composition, is equipped with agriculturally acceptable carrier and bacteria protectant composition; Wherein effective active composition is 1.0 ~ 30.0 × 10
8individual spore/gram, bacteria protectant content is 0.05 ~ 1.8%(w/w).
Trichoderma atroviride microbial inoculum of the present invention, its thalline is Trichoderma atroviride conidium, mycelia and chlamydosporic mixture; Agriculturally acceptable carrier is: diatomite, calcium carbonate, water, wheat-flour, wood chip, kaolin, one or more mixture of unsaturated hydro carbons; Described bacteria protectant is xanthan gum, sucrose, Sodium salts humic acids, one or more mixture of potassium fulvate.
The Trichoderma atroviride microbial inoculum that the present invention is more preferably, is characterized in that it is made up of following compositions:
Trichoderma atroviride Tr1208 bacteria containing amount is 1.0 ~ 30.0 × 10
8individual spore/gram,
Xanthan gum 0.05 ~ 0.1%(w/w),
Sucrose 1 ~ 1.5%(w/w),
Sodium salts humic acids or potassium fulvate 0.05 ~ 0.2%(w/w)
The present invention further discloses the preparation method of Trichoderma atroviride microbial inoculum, it is characterized in that being undertaken by following step:
1) actication of culture: preserving number is the Trichoderma atroviride Tr1208(of CGMCCNo.6828
trichoderma atroviride) bacterial strain in potato dextrose agar, activating 72h, for subsequent use;
Described potato dextrose agar (being called for short PDA substratum) consists of: potato 300 grams, glucose 20 grams, 15 ~ 20 grams, agar, 1000 milliliters, tap water, natural pH;
2) seed liquor preparation: the Trichoderma atroviride Tr1208 bacterial strain after activation, at 23 ~ 28 DEG C, wheat-flour nutrient solution or Richard's nutrient solution concussion cultivation 72 ~ 96h, obtain Trichoderma atroviride Tr1208 seed liquor after cultivation;
Described wheat-flour nutrient solution consists of: whole wheat flour 20g, glucose 20g, yeast extract paste 2g, water 1000mL, pH6.8 ~ 7.2;
Richard's nutrient solution refers to: saltpetre 10g, potassium primary phosphate 5g, magnesium sulfate 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH6.8 ~ 7.2;
3) solid fermentation is cultivated: in solid fermentation culture base-material, add water 20 ~ 45%(w/w), initial pH is between 6.5 ~ 7.5, high-temperature sterilization, then treat that temperature drops to less than 30 DEG C, access step 2) seed liquor, be 20 ~ 30 DEG C in temperature again, interval 24 ~ 30h, the condition bottom fermentation of periodic agitation cultivates 96 ~ 148h;
Described solid fermentation culture base-material (w/w) consists of: flour 2 ~ 4%, wheat bran 12 ~ 18%, waste material of edible mushroom slag or/and wood chip 77.1 ~ 84%, calcium superphosphate 0.5 ~ 1%, urea 0.2 ~ 0.5%, sucrose 0.2 ~ 0.5%, profit water after sterilizing for subsequent use;
4) measure the quantity of thalline in fermentation culture base-material, treat that fermentation base-material miospore amount reaches 10
9stop fermentation culture after more than cfu/g, often temperature is dry to cultivate material, becomes the female powder of Trichoderma atroviride after adopting Universalpulverizer to pulverize;
5) Trichoderma atroviride microbial inoculum is prepared: with bacteria containing amount for 2.0 ~ 60.0 × 10
8the female powder of individual spore/gram Trichoderma atroviride Tr1208, xanthan gum 0.05 ~ 0.1%, sucrose 1 ~ 1.5%, Sodium salts humic acids or potassium fulvate 0.05 ~ 0.2%, remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number=1:2 ~ 5, are mixed with Trichoderma atroviride microbial inoculum.
The present invention also discloses Trichoderma atroviride Tr1208(simultaneously
trichoderma atroviride) bacterial strain preparation as the application in prevention and control vegetable fungi disease microbial inoculum.Preferably as the application in control leek gray mold, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class damping-off, melon and fruit class blight, melon and fruit class root rot microbial inoculum and as the application in Promoting plant growth preparation.
The present invention can make various formulation, and as wettable powder, dust agent, granule and suspension agent etc., can also add other effective active composition carries out composite and synergistic agent, stablizer, to improve drug effect and the practicality agriculturally of preparation.
The present invention selects calcium carbonate to be carrier, and the diameter of calcium carbonate granule is 0.074mm.
The present invention selects diatomite to be carrier, and the diameter of diatomite particle is 0.074mm.
Described potato dextrose agar (being called for short PDA substratum) consists of: potato 300 grams, glucose 20 grams, 15 ~ 20 grams, agar, 1000 milliliters, tap water, natural pH;
Wheat-flour nutrient solution consists of: whole wheat flour 20g, glucose 20g, yeast extract paste 2g, add water 1000mL, pH6.8 ~ 7.2.
The application process that the invention provides Trichoderma atroviride microbial inoculum control vegetable disease is: microbial inoculum is watered 500 ~ 1000 times of liquid, in vegetable field upper part even spraying, or filling with root in plant uses, the method that soil also can be adopted to spread fertilizer over the fields carries out soil treatment, to reach the object controlling vegetable disease, effectively can prevent and treat leek gray mold, graw mold of tomato, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class blight, melon and fruit class root rot preventive effect reach 70% ~ 89%, and have obvious growth promoting effects effect to plant-growth.
The positively effect that Trichoderma atroviride Trichoderma atroviride microbial inoculum disclosed by the invention is compared with prior art had is:
(1) Trichoderma atroviride microbial inoculum disclosed by the invention has better effects to vegetable fungi disease, and compared with prior art preventive effect significantly improves;
(2) Trichoderma atroviride microbial inoculum disclosed by the invention effectively can prevent and treat leek gray mold, graw mold of tomato, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class blight, melon and fruit class root rot, and prevention and treatment range increases to some extent;
(3) Trichoderma atroviride microbial inoculum disclosed by the invention has plant-growth and significantly lures anti-and growth promoting effects effect.
Embodiment
Substantive distinguishing features of the present invention can be embodied from the following examples, but these embodiments are only as explanation, instead of limit the invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, scope of the present invention and essence are limited by claim, illustrate preparation method and the application of Trichoderma atroviride strain of the present invention below by example further.Experimental technique in following embodiment and reagent, if no special instructions, be ordinary method and all have commercially available.
Embodiment 1:
The screening and separating of Trichoderma atroviride and qualification.
Antagonistic Trichoderma screening is carried out by plate opposite culture, find that Trichoderma atroviride Tr1208 has stronger antagonistic action to pathogenic fungies such as leek gray mold, graw mold of tomato, Cucumber Target Leaf Spot, early blight of tomato, melon and fruit class blight, melon and fruit class root rot, it has stronger restraining effect to the multiple pathogenic bacteria of vegetables after testing.
Table 1, Trichoderma atroviride Tr1208 are to the fungistatic effect of leek ash arrhizus bacteria, cucumber aphid, early blight of tomato, melon and fruit class blight, melon and fruit class root rot
Trichoderma strain Tr1208 grows rapidly on PDA substratum, and in bacterium colony 3 days, growth diameter is greater than 90mm, and hyphal surface is loose, smooth, and growth radially; Cultivate 3 days visible obviously spores to produce, be initially light green, after increase gradually, in deep green, concentric wheel stripe shape.
Microscopic examination, trichoderma strain Tr1208 mycelium is colourless, branch is complicated, and tool is separated; Conidium is without barrier film, spherical in shape, and poly-head arrangement, Dan Sheng, cell walls exists obvious and small coarse projection.
With trichoderma strain Tr1208 genomic dna for template, utilize Trichoderma translation elongation factor to increase, adopt forward primer: EF1-728F(5 '-CATCGAGAAGTTCGAGAAGG-3 ') SEQ ID NO:1,
Reverse primer: TEF1 rev (5 '-GCC ATC CTT GGA GAT ACC AGC-3 ') SEQ ID NO:2; By Shanghai, bio-engineering corporation synthesizes.
Utilize primer to carry out pcr amplification, amplification translation elongation factor obtains specific band, PCR primer is checked order, and carries out BLAST comparison to sequencing result.Bacterial strain Tr1208 expanding fragment length 523bp, by comparing at GeneBank GenBank, with Trichoderma atroviride (
trichoderma atroviride) similarity be 100%.Accordingly, in conjunction with its morphological specificity and the sequencing results, bacterial strain Tr1208 is accredited as Trichoderma atroviride (
trichoderma atroviride).Proof test is inoculated by wound, Trichoderma atroviride Tr1208 does not all cause tomato, cucumber, leek and tobacco are wilted or suddenly arrive, have no its root and leaf portion generation pathology, plant strain growth is normal, illustrate that screened Trichoderma atroviride Tr1208 is safe to plant such as tomato, cucumber, leek, no pathogenicity.
Embodiment 2:
The fermentation of Trichoderma atroviride Tr1208 is standby.
1) actication of culture: preserving number is the Trichoderma atroviride Tr1208(of CGMCCNo.6828
trichoderma atroviride) bacterial strain activate in potato sucrose nutrient agar, 25 DEG C cultivate 72 ~ 96h, for subsequent use;
2) seed liquor preparation: the Trichoderma atroviride Tr1208 bacterial strain after activation, at 23 ~ 28 DEG C, wheat-flour nutrient solution concussion cultivation 72 ~ 96h, the Trichoderma atroviride Tr1208 seed liquor of gained after cultivating;
3) solid fermentation is cultivated: the initial pH of solid fermentation culture base-material is between 6.5 ~ 7.5, high-temperature sterilization, treats that temperature drops to less than 30 DEG C, access step 2) seed liquor, be 20 ~ 30 DEG C in temperature again, the condition bottom fermentation of periodic agitation (interval 24 ~ 30h) cultivates 96 ~ 148h.
Described solid fermentation culture base-material formula one (w/w) consists of: flour 2%, wheat bran 12%, waste material of edible mushroom slag (or/and wood chip) 84%, calcium superphosphate 1%, urea 0.5%, sucrose 0.5%, what add that water accounts for total base-material (w/w) consists of 20%, and rear sterilizing is for subsequent use;
Formula the consisting of of two (w/w): flour 3%, wheat bran 15%, waste material of edible mushroom slag (or/and wood chip) 80.6%, calcium superphosphate 0.75%, urea 0.35%, sucrose 0.3%, what add that water accounts for total base-material (w/w) consists of 37.5%, and rear sterilizing is for subsequent use;
Formula the consisting of of two (w/w): flour 4%, wheat bran 18%, waste material of edible mushroom slag (or/and wood chip) 77.1%, calcium superphosphate 0.5%, urea 0.2%, sucrose 0.2%, what add that water accounts for total base-material (w/w) consists of 45%, and rear sterilizing is for subsequent use.
4) measure the quantity of thalline in fermentation culture base-material, treat that fermentation base-material miospore amount reaches 10
9stop fermentation culture after more than cfu/g, often temperature is dry to cultivate material, obtains the female powder of Trichoderma atroviride after adopting Universalpulverizer to pulverize.
Table 2, different times Trichoderma atroviride Tr208 spore population density change (solid fermentation substratum)
Embodiment 3:
Trichoderma atroviride microbial inoculum, is characterized in that it is made up of following compositions: the Trichoderma atroviride Tr1208 bacteria containing amount that preserving number is is 1.0 × 10
8individual spore/gram (preparation method is with embodiment 2),
Xanthan gum 0.05%,
Sucrose 1%,
Sodium salts humic acids or potassium fulvate 0.05%,
Remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number=1:2.
Embodiment 4:
Trichoderma atroviride microbial inoculum, is characterized in that it is made up of following compositions: Trichoderma atroviride Tr1208 bacteria containing amount is 10.0 × 10
8individual spore/gram (preparation method is with embodiment 2),
Xanthan gum 0.1%(w/w),
Sucrose 1%(w/w),
Potassium fulvate 0.2%(w/w),
Remaining amount is wheat-flour and kaolin, wherein wheat-flour: kaolinic ratio of weight and number is 1:5.
Embodiment 5:
Trichoderma atroviride microbial inoculum, is characterized in that it is made up of following compositions: Trichoderma atroviride Tr1208 bacteria containing amount is 20.0 × 10
8individual spore/gram (preparation method is with embodiment 2),
Xanthan gum 0.05%(w/w),
Sucrose 1.5%(w/w),
Sodium salts humic acids or potassium fulvate 0.05%(w/w)
Remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number is 1:3.
Embodiment 6:
Trichoderma atroviride microbial inoculum, is characterized in that it is made up of following compositions: Trichoderma atroviride Tr1208 bacteria containing amount is 30.0 × 10
8individual spore/gram (preparation method is with embodiment 2),
Xanthan gum 0.08%(w/w),
Sucrose 1.2%(w/w),
Sodium salts humic acids or potassium fulvate 0.08%(w/w)
Remaining amount is calcium carbonate and diatomite support, wherein wood chip: diatomaceous ratio of weight and number is 1:4.
Embodiment 7:
Trichoderma atroviride microbial inoculum, is characterized in that it is made up of following compositions: Trichoderma atroviride Tr1208 bacteria containing amount is 30.0 × 10
8individual spore/gram (preparation method is with embodiment 2),
Xanthan gum 0.1%(w/w),
Sucrose 1.5%(w/w),
Sodium salts humic acids or potassium fulvate 0.2%(w/w)
Remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number is 1:5.
Embodiment 8
The preparation method of Trichoderma atroviride microbial inoculum:
1) actication of culture: preserving number is the Trichoderma atroviride Tr1208(of CGMCCNo.6828
trichoderma atroviride) bacterial strain in potato dextrose agar, activate 72h, for subsequent use;
Described potato dextrose agar (being called for short PDA substratum) consists of: potato 300 grams, glucose 20 grams, 15 grams, agar, 1000 milliliters, tap water, natural pH;
2) seed liquor preparation: the Trichoderma atroviride Tr1208 bacterial strain after activation, at 25 DEG C, wheat-flour nutrient solution 72h, obtains Trichoderma atroviride Tr1208 seed liquor after cultivation;
Described wheat-flour nutrient solution consists of: whole wheat flour 20g, glucose 20g, yeast extract paste 2g, water 1000mL, pH6.8;
Richard's nutrient solution consists of: saltpetre 10g, potassium primary phosphate 5g, magnesium sulfate 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH6.8;
3) solid fermentation is cultivated: in solid fermentation culture base-material, add water 35%(w/w), initial pH is between 6.5, high-temperature sterilization, then treat that temperature drops to less than 30 DEG C, access step 2) seed liquor, be 25 DEG C in temperature again, interval 24 ~ 30h, the condition bottom fermentation of periodic agitation cultivates 96 ~ 148h;
Described solid fermentation culture base-material (w/w) consists of: flour 4%, wheat bran 12%, waste material of edible mushroom slag or/and wood chip 77.1%, calcium superphosphate 0.5%, urea 0.3 %, sucrose 0.3%, profit water after sterilizing for subsequent use;
4) measure the quantity of thalline in fermentation culture base-material, treat that fermentation base-material miospore amount reaches 10
9stop fermentation culture after more than cfu/g, often temperature is dry to cultivate material, adopt Universalpulverizer to pulverize, sieve after become the female powder of Trichoderma atroviride;
5) Trichoderma atroviride microbial inoculum is prepared: with bacteria containing amount for 2.0 ~ 60.0 × 10
8the female powder of individual spore/gram Trichoderma atroviride Tr1208, xanthan gum 0.08%, sucrose 1.2%, Sodium salts humic acids or potassium fulvate 0.08%, remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number=1:2, is mixed with Trichoderma atroviride microbial inoculum.
Embodiment 9
The preparation method of Trichoderma atroviride microbial inoculum:
1) actication of culture: preserving number is the Trichoderma atroviride Tr1208(of CGMCCNo.6828
trichoderma atroviride) bacterial strain in potato dextrose agar, activate 72h, for subsequent use;
Described potato dextrose agar (being called for short PDA substratum) consists of: potato 300 grams, glucose 20 grams, 20 grams, agar, 1000 milliliters, tap water, natural pH;
2) seed liquor preparation: the Trichoderma atroviride Tr1208 bacterial strain after activation, at 23 DEG C, 75h is cultivated in the concussion of Richard's nutrient solution, obtains Trichoderma atroviride Tr1208 seed liquor after cultivation;
Richard's nutrient solution consists of: saltpetre 10g, potassium primary phosphate 5g, magnesium sulfate 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH7.2;
3) solid fermentation is cultivated: in solid fermentation culture base-material, add water 45%(w/w), initial pH is between 7.5, high-temperature sterilization, then treat that temperature drops to less than 30 DEG C, access step 2) seed liquor, be 30 DEG C in temperature again, interval 24 ~ 30h, the condition bottom fermentation of periodic agitation cultivates 96 ~ 148h;
Described solid fermentation culture base-material (w/w) consists of: flour 4%, wheat bran 18%, waste material of edible mushroom slag or/and wood chip 84%, calcium superphosphate 1%, urea 0.5%, sucrose 0.5%, profit water after sterilizing for subsequent use;
4) measure the quantity of thalline in fermentation culture base-material, treat that fermentation base-material miospore amount reaches 10
9stop fermentation culture after more than cfu/g, often temperature is dry to cultivate material, adopt Universalpulverizer to pulverize, sieve after become the female powder of Trichoderma atroviride;
5) Trichoderma atroviride microbial inoculum is prepared: with bacteria containing amount for 2.0 ~ 60.0 × 10
8the female powder of individual spore/gram Trichoderma atroviride Tr1208, xanthan gum 0.1%, sucrose 1.5%, Sodium salts humic acids or potassium fulvate 0.2%, remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number=1:2 ~ 5, are mixed with Trichoderma atroviride microbial inoculum.
Embodiment 10:
The protection effect of Trichoderma atroviride microbial inoculum:
Carry out the pot experiment of preventing and treating leek gray mold to developed Trichoderma atroviride microbial inoculum, test-results is in table 3.Leek gray mold results from pot experiment test shows, Trichoderma atroviride microbial inoculum has good prevention and control effect to leek gray mold, the sickness rate of contrast is 23.5%, the process morbidity of Trichoderma atroviride is lighter, sickness rate is between 4.7% ~ 5.5%, and date of the onset comparatively contrasts postponement 3 ~ 5d, after Trichoderma atroviride 500,1000 times process, preventive effect is respectively 80.0% and 76.6%, and observation has certain growth promoting effect.
Table 3, Trichoderma atroviride Tr1208 prevent and treat leek gray mold
Carry out the pot experiment of preventing and treating graw mold of tomato to developed Trichoderma atroviride microbial inoculum, test-results is in table 4.Graw mold of tomato results from pot experiment test shows, Trichoderma atroviride microbial inoculum has good prevention and control effect to graw mold of tomato, the sickness rate of contrast is 10.5%, the process morbidity of Trichoderma atroviride is lighter, sickness rate is between 1.10% ~ 1.93%, and date of the onset comparatively contrasts postponement 3 ~ 6d, after Trichoderma atroviride 500,1000 times process, preventive effect is respectively 89.5% and 81.6%, and observation has good growth promoting effects effect to the growth of tomato.
Table 4, Trichoderma atroviride Tr1208 prevent and treat graw mold of tomato
In addition, we have also carried out the test of pesticide effectiveness for cucumber rhizoctonia rot, cucumber downy mildew, powdery mildew of cucumber, the results are shown in Table 5 ~ 8.Wherein, Trichoderma atroviride microbial inoculum root irrigation in seedling stage control cucumber rhizoctonia rot, test of pesticide effectiveness result shows, without significant difference between Trichoderma atroviride microbial inoculum and 70% thiophanate_methyl WP process, each process prevention effect is between 72.59 ~ 74.66%.Test discovery, Trichoderma atroviride Tr1208 different treatment all can promote that cucumber plant grows, and plant height comparatively contrasts raising 14.88% ~ 18.19%, and leaf look dark green simultaneously, and result shows that its short long-acting fruit is comparatively obvious.
Table 5, Trichoderma atroviride Tr1208 root irrigation in the seedling stage control cucumber rhizoctonia rot test of pesticide effectiveness
Table 6, Trichoderma atroviride Tr1208 root irrigation in the seedling stage control cucumber rhizoctonia rot test of pesticide effectiveness
Trichoderma atroviride Tr1208 sprays, and process prevents and treats cucumber downy mildew, Powdery Mildew all has good prevention effect, and its 500 times of liquid process are suitable with the preventive effect of current conventional medicament 80% zinc manganese ethylenebisdithiocarbamate WP.
Table 7, Trichoderma atroviride Tr1208 prevent and treat the cucumber downy mildew test of pesticide effectiveness
Table 8, Trichoderma atroviride Tr1208 prevent and treat the powdery mildew of cucumber test of pesticide effectiveness
After adopting Trichoderma atroviride Tr1208 tri-root irrigations after being transplanted by tomato, investigate it and 65.5% is reached to the prevention effect of root knot nematode, the root knot quantity of tomato root can be reduced, show with Trichoderma atroviride control root knot nematode be have certain effect.Infect after the tomato of root knot nematode uses Trichoderma atroviride and can reduce its root root knot number, strengthen improving activity of root system, extend productive phase.
SEQUENCE LISTING
<110> Tianjin Plant Protection Institute
Trichoderma atroviride preparation of a <120> prevention and control vegetable fungi disease and preparation method thereof
<160> 2
<170> PatentIn version 3.5
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<212> DNA
<213> artificial sequence
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catcgagaag ttcgagaagg 20
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<212> DNA
<213> artificial sequence
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gccatccttg gagataccag c 21
Claims (4)
1. a preparation method for Trichoderma atroviride microbial inoculum, is characterized in that being undertaken by following step:
1) actication of culture: preserving number is the Trichoderma atroviride Tr1208(of CGMCCNo.6828
trichoderma atroviride) bacterial strain in potato dextrose agar, activate 72h, for subsequent use;
Described potato dextrose agar, is called for short PDA substratum and consists of: potato 300 grams, glucose 20 grams, 15 ~ 20 grams, agar, 1000 milliliters, tap water, natural pH;
2) seed liquor preparation: the preserving number after activation is the Trichoderma atroviride Tr1208 bacterial strain of CGMCCNo.6828, at 23 ~ 28 DEG C, wheat-flour nutrient solution or Richard's nutrient solution concussion cultivation 72 ~ 96h, obtain Trichoderma atroviride Tr1208 seed liquor after cultivation;
Described wheat-flour nutrient solution consists of: whole wheat flour 20g, glucose 20g, yeast extract paste 2g, water 1000mL, pH6.8 ~ 7.2;
Richard's nutrient solution consists of: saltpetre 10g, potassium primary phosphate 5g, magnesium sulfate 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH6.8 ~ 7.2;
3) solid fermentation is cultivated: in solid fermentation culture base-material, add water 20 ~ 45%(w/w), initial pH is between 6.5 ~ 7.5, high-temperature sterilization, then treat that temperature drops to less than 30 DEG C, access step 2) seed liquor, be 20 ~ 30 DEG C in temperature again, interval 24 ~ 30h, the condition bottom fermentation of periodic agitation cultivates 96 ~ 148h;
Described solid fermentation culture base-material (w/w) consists of: flour 2 ~ 4%, wheat bran 12 ~ 18%, waste material of edible mushroom slag or/and wood chip 77.1 ~ 84%, calcium superphosphate 0.5 ~ 1%, urea 0.2 ~ 0.5%, sucrose 0.2 ~ 0.5%, profit water after sterilizing for subsequent use;
4) measure the quantity of thalline in fermentation culture base-material, treat that fermentation base-material miospore amount reaches 10
9stop fermentation culture after more than cfu/g, often temperature is dry to cultivate material, adopt Universalpulverizer to pulverize, sieve after become the female powder of Trichoderma atroviride;
5) Trichoderma atroviride microbial inoculum is prepared: with bacteria containing amount for 1.0 ~ 30.0 × 10
8individual spore/gram Trichoderma atroviride, preserving number is the female powder of Tr1208 of CGMCCNo.6828, xanthan gum 0.05 ~ 0.1%, sucrose 1 ~ 1.5%, Sodium salts humic acids or potassium fulvate 0.05 ~ 0.2%, remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number=1:2 ~ 5, are mixed with Trichoderma atroviride microbial inoculum.
2. the Trichoderma atroviride microbial inoculum that obtains of the preparation method of Trichoderma atroviride microbial inoculum according to claim 1, is characterized in that it is made up of following compositions:
Preserving number is the Trichoderma atroviride Tr1208 bacteria containing amount of CGMCCNo.6828 is 1.0 ~ 30.0 × 10
8individual spore/gram,
Xanthan gum 0.05 ~ 0.1%(w/w),
Sucrose 1 ~ 1.5%(w/w),
Sodium salts humic acids or potassium fulvate 0.05 ~ 0.2%(w/w)
Remaining amount is calcium carbonate and diatomite support, wherein calcium carbonate: diatomaceous ratio of weight and number is 1:2 ~ 5.
3. Trichoderma atroviride microbial inoculum described in claim 2 is being prepared as the application in control leek gray mold, Cucumber Target Leaf Spot, powdery mildew of cucumber, cucumber downy mildew, early blight of tomato, melon and fruit class damping-off, melon and fruit class blight, melon and fruit class root rot microbial inoculum.
4. Trichoderma atroviride microbial inoculum described in claim 2 is being prepared as the application in Promoting plant growth microbial inoculum.
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