CN103484384A

CN103484384A - Trichoderma atroviride preparation for preventing and controlling vegetable fungal diseases and preparation method of trichoderma atroviride preparation thereof

Info

Publication number: CN103484384A
Application number: CN201310462539.4A
Authority: CN
Inventors: 王勇; 高苇; 王万立; 张春祥; 郝永娟; 刘春艳; 霍建飞
Original assignee: TIANJIN PLANT PROTECTION INSTITUTE
Current assignee: Tianjin Academy of Agricultural Sciences
Priority date: 2013-10-08
Filing date: 2013-10-08
Publication date: 2014-01-01
Anticipated expiration: 2033-10-08
Also published as: CN103484384B

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

Dark green wooden removing mildew of a kind of prevention and control vegetable fungi disease and preparation method thereof

Technical field

The invention belongs to the microbial pesticide technical field, be specifically related to a kind of dark green wooden removing mildew and preparation method thereof and application of antagonism vegetables pathogenic fungi.

Background technology

Along with the quickening of China Agricultural Structure Adjustment paces and the raising of living standards of the people, the demand of the variation of vegetable variety and anniversary stable market supply strengthens day by day, the scale that China vegetables produce constantly enlarges, output grows steadily, and has become in China's plant husbandry the second largest farm crop that are 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 the development along with China's vegetables production, the diversity of cultivation mode, vegetable growing kind and kind rich, especially the variation of planting environment, cause some Minor diseases to rise, the pathogenic bacteria host range enlarges, and has produced many urgently new diseases of research, some the old and new's diseases alternately cause harm, and these are all seriously restricting the development of China's vegetables industry.Fungal diseases 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 wherein, all occur comparatively serious, cause Severe Reduction, even total crop failure, become the restrictive barriers that vegetables produce.At present such disease of control still mainly adopts chemical agent, and usually fresh out of medicine sales of just gathering, not only cause the vegetables pollution, also very easily causes the resistance problem simultaneously.Therefore research and develop safety and environment mutually harmonious microbial pesticide become inevitable, wooden mould this class beneficial microorganism wherein, 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, 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 prevent and treat fungal disease.The mould research aspect biological and ecological methods to prevent plant disease, pests, and erosion of relevant wood has up to now been carried out nearly 70 years, commercial wooden removing mildew occurred abroad.As with control fruit trees in plum line silver leaf ( chondrostereum purpureum) the commercialization production in West Europe of wooden removing mildew; The exploitation of the Makhteshim Agan company of Israel take the biological prevention and control agent that trichoderma harziarum T39 bacterial strain is fermented liquid, trade(brand)name Trichodex, but formulation is 25% wet-milling, it can for prevent and treat ash arrhizus bacteria ( botrytis cinerea), the sclerotium germ ( scleratina sclerotiorum), leaf mold ( cladosporium fulvum) etc. disease, also have at home the research and utilization Trichoderma to control Plant diseases.21 century is called as environmental protection century, and the application of chemical agent progressively is restricted, and therefore the biological control research nature of vegetable disease is called to hot issue.

The present invention just is being based on above reason, with current vegetables, cause harm comparatively serious, lack again the 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, find the microorganism with efficient antagonistic activity from Microbial resources, intend finding new resource for research and development new bio microbial inoculum.

Summary of the invention

The present invention aims to provide a kind of dark green wooden removing mildew of preventing and treating vegetable disease and preparation method thereof, can, for preventing and treating vegetable fungi disease, solve pesticide residue, resistance and problem of environmental pollution that chemical pesticide control brings.The present invention can be used for preventing and treating 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., the vegetables microbiobacterial agent that have efficient, spectrum, stable performance, is produced on a large scale.For realizing this purpose, the invention discloses following technology contents:

One strain has the dark green trichoderma strain of antagonism vegetables pathogenic fungi activity, it is characterized in that this bacterial strain is dark green trichoderma Tr1208(Trichoderma atroviride), in preservation China Committee for Culture Collection of Microorganisms common micro-organisms on November 15 center in 2012, preserving number was CGMCCNo.6828.Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.

The dark green trichoderma strain Tr1208(of preservation of the present invention trichoderma atroviride) physiology, biochemical characteristic as follows:

Trichoderma strain Tr1208 grows rapidly on the PDA flat board, the flat board of bacterium colony covering diameter 90mm in 3 days, and radially, surface porosity is smooth; Sporulation was obviously arranged as seen in the 3rd day what cultivate, be initially light green, increase gradually later, present deep green, the gonimoblast bundle is arranged in concentric wheel line.The mycelium that microscopic examination is cultivated is colourless, branch is complicated, the branch mycelia with tabula, consists of; The conidium produced is without barrier film, spherical in shape, and be polymerized to head and arrange, Dan Sheng, there is obvious and small coarse projection in cell walls.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 dark green trichoderma, it is characterized in that it is the dark green trichoderma Tr1208(that is CGMCCNo.6828 by preserving number trichoderma atroviride) secondary metabolite of thalline and it is the effective active composition, be equipped with the upper acceptable carrier of agricultural and thalline protective material and form; Wherein the effective active composition is 1.0 ~ 30.0 * 10 ⁸individual spore/gram, thalline protective material content is 0.05 ~ 1.8%(w/w).

The preferred dark green trichoderma of the present invention, is characterized in that it is the dark green trichoderma Tr1208(that is CGMCCNo.6828 by preserving number trichoderma atroviride) thalline is the effective active composition, be equipped with the upper acceptable carrier of agricultural and thalline protective material and form; Wherein the effective active composition is 1.0 ~ 30.0 * 10 ⁸individual spore/gram, thalline protective material content is 0.05 ~ 1.8%(w/w).

Dark green trichoderma of the present invention, its thalline is dark green trichoderma conidium, mycelia and chlamydosporic mixture; On agricultural, acceptable carrier is: one or more mixture of diatomite, calcium carbonate, water, wheat-flour, wood chip, kaolin, unsaturated hydro carbons; Described thalline protective material is one or more mixture of xanthan gum, sucrose, Sodium salts humic acids, potassium fulvate.

The dark green trichoderma that the present invention is more preferably is characterized in that it is comprised of following compositions:

Dark green trichoderma 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 dark green trichoderma, it is characterized in that being undertaken by following step:

1) actication of culture: the dark green trichoderma Tr1208(that preserving number is CGMCCNo.6828 trichoderma atroviride) bacterial strain activate 72h in potato dextrose agar, standby;

Described potato dextrose agar (be called for short PDA substratum) consists of: 1000 milliliters of potato 300 grams, glucose 20 grams, agar 15 ~ 20 grams, tap water, natural pH;

2) seed liquor preparation: the dark green trichoderma Tr1208 bacterial strain after activation, under 23 ~ 28 ℃, 72 ~ 96h is cultivated in wheat-flour nutrient solution or the concussion of Richard's nutrient solution, obtains dark green trichoderma 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, sal epsom 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH6.8～7.2;

3) solid fermentation is cultivated: add water 20 ~ 45%(w/w in the solid fermentation culture base-material), initial pH is between 6.5 ~ 7.5, high-temperature sterilization, then treat that temperature drops to below 30 ℃, access step 2) seed liquor, in temperature, be 20 ~ 30 ℃ again, interval 24 ~ 30h, the condition bottom fermentation regularly stirred is cultivated 96 ~ 148h;

Described solid fermentation culture base-material (w/w) consists of: flour 2 ~ 4%, and wheat bran 12 ~ 18%, the waste material of edible mushroom slag is or/and wood chip 77.1 ~ 84%, calcium superphosphate 0.5 ~ 1%, urea 0.2 ~ 0.5%, sucrose 0.2 ~ 0.5%, after profit water, sterilizing is standby;

4) measure the quantity of thalline in the fermentation culture base-material, base-material miospore amount to be fermented reaches 10 ⁹stop fermentation culture after cfu/g is above, cultivate material frequent warm dry, adopt Universalpulverizer to pulverize the female powder of the dark green trichoderma of rear one-tenth;

5) prepare dark green trichoderma: take bacteria containing amount as 2.0 ~ 60.0 * 10 ⁸the female powder of the dark green trichoderma Tr1208 of individual spore/gram, 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 dark green trichoderma.

The present invention also discloses dark green trichoderma Tr1208(simultaneously trichoderma atroviride) bacterial strain the preparation as the application in prevention and control vegetable fungi disease microbial inoculum.Preferably in the application as 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 the Promoting plant growth preparation.

The present invention can make various formulations, as wettable powder, dust agent, granule and suspension agent etc., can also add other effective active composition to carry out composite and synergistic agent, stablizer, with the drug effect that improves preparation and the practicality on agricultural.

It is carrier that the present invention selects calcium carbonate, and the diameter of calcium carbonate granule is 0.074mm.

It is carrier that the present invention selects diatomite, and the diameter of diatomite particle is 0.074mm.

The 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 dark green trichoderma control vegetable disease is: microbial inoculum is watered to 500 ~ 1000 times of liquid, in vegetables over-ground part even spraying, or fill with root in plant and use, the method that also can adopt soil to spread fertilizer over the fields is carried out soil treatment, to reach the purpose of controlling vegetable disease, can effectively 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 reaches 70% ~ 89%, and plant-growth is had to obvious promotion growth.

The positively effect that the dark green trichoderma of dark green trichoderma disclosed by the invention compared with prior art had is:

(1) dark green trichoderma disclosed by the invention has better effects to vegetable fungi disease, and compared with prior art preventive effect obviously improves;

(2) dark green trichoderma disclosed by the invention can effectively be prevented and treated 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) dark green trichoderma disclosed by the invention has and significantly lures anti-and promotion growth plant-growth.

Embodiment

Substantive distinguishing features of the present invention can be embodied from the following examples, but these embodiment are only as explanation, rather than limit the invention, one of skill in the art can make improvements and change according to spirit of the present invention, described these improvement and variation all should be considered as within the scope of the invention, scope of the present invention and essence are limited by claim, further illustrate preparation method and the application of the dark green trichoderma strain of the present invention below by example.Experimental technique in following embodiment and reagent, if no special instructions, be ordinary method and all have commercially available.

Embodiment 1:

Screening and separating and the evaluation of dark green trichoderma.

Stand facing each other to cultivate by plate and carry out the Antagonistic Trichoderma screening, find that dark green trichoderma 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, the dark green trichoderma Tr1208 fungistatic effect to leek ash arrhizus bacteria, Cucumber Target Leaf Spot bacterium, early blight of tomato, melon and fruit class blight, melon and fruit class root rot

Figure 2013104625394100002DEST_PATH_IMAGE001

Trichoderma strain Tr1208 grows rapidly on the PDA substratum, and in bacterium colony 3 days, growth diameter is greater than 90mm, mycelia surface porosity, smooth, and growth is radially; Cultivate 3 days visible obviously Sporulations, be initially light green, after increase gradually, be 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 is arranged, Dan Sheng, and there is obvious and small coarse projection in cell walls.

The trichoderma strain Tr1208 genomic dna of take is template, utilizes the Trichoderma translation elongation factor to be increased, and adopts 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; Synthetic by Shanghai bio-engineering corporation.

Utilize primer to carry out pcr amplification, the amplification translation elongation factor obtains specific band, by the order-checking of PCR product, and sequencing result is carried out to the BLAST comparison.Bacterial strain Tr1208 expanding fragment length 523bp, by comparing at the GeneBank GenBank, with dark green trichoderma ( trichoderma atroviride) similarity be 100%.Accordingly, in conjunction with its morphological specificity and the sequencing results, by bacterial strain Tr1208 be accredited as dark green trichoderma ( trichoderma atroviride).Inoculate proof test by wound, dark green trichoderma Tr1208 does not all cause tomato, cucumber, leek and tobacco wilting or sudden arriving, have no its root and leaf section pathology occurs, plant strain growth is normal, illustrate that screened dark green trichoderma Tr1208 is safe to plant such as tomato, cucumber, leek, no pathogenicity.

Embodiment 2:

The fermentation preparation of dark green trichoderma Tr1208.

1) actication of culture: the dark green trichoderma Tr1208(that preserving number is CGMCCNo.6828 trichoderma atroviride) bacterial strain in the potato sucrose nutrient agar, activate, cultivate 72 ~ 96h for 25 ℃, standby;

2) seed liquor preparation: the dark green trichoderma Tr1208 bacterial strain after activation, under 23 ~ 28 ℃, 72 ~ 96h is cultivated in the concussion of wheat-flour nutrient solution, the dark green trichoderma 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, treat that temperature drops to below 30 ℃, the access step 2) seed liquor, be 20 ~ 30 ℃ in temperature again, regularly stir the condition bottom fermentation of (interval 24 ~ 30h) and cultivate 96 ~ 148h.

Consisting of of described solid fermentation culture base-material formula one (w/w): 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 water to account for total base-material (w/w) consists of 20%, and rear sterilizing is standby;

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 water to account for total base-material (w/w) consists of 37.5%, and rear sterilizing is standby;

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 water to account for total base-material (w/w) consists of 45%, and rear sterilizing is standby.

4) measure the quantity of thalline in the fermentation culture base-material, base-material miospore amount to be fermented reaches 10 ⁹stop fermentation culture after cfu/g is above, cultivate the frequent temperature drying of material, adopt Universalpulverizer to pulverize the rear dark green trichoderma mother powder that obtains.

Table 2, the dark green trichoderma Tr208 of different times spore population density change (solid fermentation substratum)

Embodiment 3:

Dark green trichoderma is characterized in that it is comprised of following compositions: the dark green trichoderma 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:

Dark green trichoderma is characterized in that it is comprised of following compositions: dark green trichoderma 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:

Dark green trichoderma is characterized in that it is comprised of following compositions: dark green trichoderma 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:

Dark green trichoderma is characterized in that it is comprised of following compositions: dark green trichoderma 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:

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 dark green trichoderma:

Described potato dextrose agar (be called for short PDA substratum) consists of: 1000 milliliters of potato 300 grams, glucose 20 grams, agar 15 grams, tap water, natural pH;

2) seed liquor preparation: the dark green trichoderma Tr1208 bacterial strain after activation, under 25 ℃, wheat-flour nutrient solution 72h, obtain dark green trichoderma 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, sal epsom 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH6.8;

3) solid fermentation is cultivated: add water 35%(w/w in the solid fermentation culture base-material), initial pH is between 6.5, high-temperature sterilization, then treat that temperature drops to below 30 ℃, access step 2) seed liquor, in temperature, be 25 ℃ again, interval 24 ~ 30h, the condition bottom fermentation regularly stirred is cultivated 96 ~ 148h;

Described solid fermentation culture base-material (w/w) consists of: flour 4%, and wheat bran 12%, the waste material of edible mushroom slag is or/and wood chip 77.1%, calcium superphosphate 0.5%, urea 0.3 %, sucrose 0.3%, after profit water, sterilizing is standby;

4) measure the quantity of thalline in the fermentation culture base-material, base-material miospore amount to be fermented reaches 10 ⁹stop fermentation culture after cfu/g is above, cultivation material often temperature is dry, adopts Universalpulverizer pulverizing, the female powder of the dark green trichoderma of the rear one-tenth that sieves;

5) prepare dark green trichoderma: take bacteria containing amount as 2.0 ~ 60.0 * 10 ⁸the female powder of the dark green trichoderma Tr1208 of individual spore/gram, 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 dark green trichoderma.

Embodiment 9

The preparation method of dark green trichoderma:

Described potato dextrose agar (be called for short PDA substratum) consists of: 1000 milliliters of potato 300 grams, glucose 20 grams, agar 20 grams, tap water, natural pH;

2) seed liquor preparation: the dark green trichoderma Tr1208 bacterial strain after activation, under 23 ℃, 75h is cultivated in the concussion of Richard's nutrient solution, obtains dark green trichoderma Tr1208 seed liquor after cultivation;

Richard's nutrient solution consists of: saltpetre 10g, potassium primary phosphate 5g, sal epsom 2.5 g, iron(ic) chloride 0.02 g, sucrose 50 g, water 1000 mL, pH7.2;

3) solid fermentation is cultivated: add water 45%(w/w in the solid fermentation culture base-material), initial pH is between 7.5, high-temperature sterilization, then treat that temperature drops to below 30 ℃, access step 2) seed liquor, in temperature, be 30 ℃ again, interval 24 ~ 30h, the condition bottom fermentation regularly stirred is cultivated 96 ~ 148h;

Described solid fermentation culture base-material (w/w) consists of: flour 4%, and wheat bran 18%, the waste material of edible mushroom slag is or/and wood chip 84%, calcium superphosphate 1%, urea 0.5%, sucrose 0.5%, after profit water, sterilizing is standby;

5) prepare dark green trichoderma: take bacteria containing amount as 2.0 ~ 60.0 * 10 ⁸the female powder of the dark green trichoderma Tr1208 of individual spore/gram, 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 dark green trichoderma.

Embodiment 10:

The protection effect of dark green trichoderma:

Developed dark green trichoderma is prevented and treated to the pot experiment of leek gray mold, test-results is in Table 3.Leek gray mold results from pot experiment test shows, have the preferably prevention and control effect of dark green trichoderma to the leek gray mold, the sickness rate of contrast is 23.5%, the processing morbidity of dark green trichoderma is lighter, sickness rate is between 4.7% ~ 5.5%, and contrast date of the onset and postpone 3 ~ 5d, after dark 500,1000 times of processing of green trichoderma, preventive effect is respectively 80.0% and 76.6%, and observation has certain growth promoting effect.

Table 3, dark green trichoderma Tr1208 control leek gray mold

Figure 2013104625394100002DEST_PATH_IMAGE003

Developed dark green trichoderma is prevented and treated to the pot experiment of graw mold of tomato, test-results is in Table 4.The graw mold of tomato results from pot experiment test shows, have the preferably prevention and control effect of dark green trichoderma to graw mold of tomato, the sickness rate of contrast is 10.5%, the processing morbidity of dark green trichoderma is lighter, sickness rate is between 1.10% ~ 1.93%, and contrast date of the onset and postpone 3 ~ 6d, after dark 500,1000 times of processing of green trichoderma, preventive effect is respectively 89.5% and 81.6%, and observes growth to tomato and have and promote preferably growth.

Table 4, dark green trichoderma Tr1208 control 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, dark green trichoderma root irrigation in seedling stage control cucumber rhizoctonia rot, test of pesticide effectiveness result shows, between dark green trichoderma and 70% thiophanate_methyl WP processing, without significant difference, each processes prevention effect between 72.59 ~ 74.66%.Test discovery simultaneously, dark green trichoderma Tr1208 different treatment all can promote the cucumber plant growth, and plant height contrasts and improves 14.88% ~ 18.19%, and the leaf look dark green, and result shows that its short long-acting fruit is comparatively obvious.

Table 5, dark green trichoderma Tr1208 root irrigation in the seedling stage control cucumber rhizoctonia rot test of pesticide effectiveness

Figure 2013104625394100002DEST_PATH_IMAGE005

Table 6, dark green trichoderma Tr1208 root irrigation in the seedling stage control cucumber rhizoctonia rot test of pesticide effectiveness

The control cucumber downy mildew is processed in dark green trichoderma Tr1208 spraying, Powdery Mildew all has prevention effect preferably, and its 500 times of liquid are processed suitable with the preventive effect of medicament 80% zinc manganese ethylenebisdithiocarbamate WP at present commonly used.

Table 7, the dark green trichoderma Tr1208 control cucumber downy mildew test of pesticide effectiveness

Table 8, dark green trichoderma Tr1208 prevent and treat the powdery mildew of cucumber test of pesticide effectiveness

Figure 2013104625394100002DEST_PATH_IMAGE002

After adopting tri-root irrigations of dark green trichoderma Tr1208 after transplanting by tomato, investigate its prevention effect to root knot nematode and reach 65.5%, can reduce the root knot quantity of tomato root, what show to control root knot nematode with dark green trichoderma is that certain effect is arranged.Can reduce its root root knot number after the tomato of infection root knot nematode is used dark green trichoderma, strengthen improving activity of root system, extend productive phase.

SEQUENCE LISTING

<110 > Tianjin Plant Protection Institute

<120 > dark green wooden removing mildew of a kind of prevention and control vegetable fungi disease and preparation method thereof

<160> 2

<170> PatentIn version 3.5

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<211> 20

<212> DNA

<213 > artificial sequence

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catcgagaag ttcgagaagg 20

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<212> DNA

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gccatccttg gagataccag c 21

Claims

1. A Trichoderma atroviride strain with the activity of antagonizing vegetable pathogenic fungi, characterized in that the strain is Trichoderma atroviride ( Trichoderma atroviride ), which has been preserved in the General Microbiology Center of the China Committee for the Preservation of Microbial Cultures, and the preservation number is CGMCC No. 6828.

2. A Trichoderma atroviride fungus agent, characterized in that it is composed of Trichoderma atroviride Tr1208 ( Trichoderma atroviride ) thallus and its secondary metabolites with preservation number CGMCCNo.6828 as effective active ingredients, together with agricultural It is composed of acceptable carrier and cell protection agent; the effective active ingredient is 1.0~30.0× ¹⁰⁸ spores/g, and the content of cell protection agent is 0.05~1.8% (w/w).

3. The dark green Trichoderma inoculum agent according to claim 2, characterized in that it is made of dark green Trichoderma Tr1208 ( Trichoderma atroviride ) thalline whose preservation number is CGMCCNo.6828 as an effective active ingredient, and is equipped with agriculturally acceptable Composed of carrier and cell protectant; the effective active ingredient is 1.0~30.0× ¹⁰⁸ spores/g, and the content of cell protectant is 0.05~1.8% (w/w).

4. the described dark green Trichoderma inoculum agent of claim 2, wherein said thalline is the mixing of Trichoderma dark green conidia, hyphae and chlamydospore; Agriculturally acceptable carrier is: diatomaceous earth, Calcium carbonate, water, wheat flour, sawdust, kaolin, unsaturated hydrocarbons or a mixture of one or more; the bacterial cell protection agent is one or more of xanthan gum, sucrose, sodium humate, potassium fulvic acid mixture.

5. the dark green Trichoderma fungus agent described in any one of claim 2-4 is characterized in that it is made up of following composition:

The bacterial content of Trichoderma dark green Tr1208 is 1.0~30.0× ¹⁰⁸ spores/g,

Xanthan gum 0.05~0.1% (w/w),

Sucrose 1~1.5% (w/w),

Sodium humate or potassium fulvate 0.05~0.2% (w/w)

The remaining amount is calcium carbonate and diatomite carrier, wherein the ratio of calcium carbonate:diatomite in parts by weight is 1:2~5.

6. the preparation method of the described Trichoderma dark green fungus agent described in claim 2-4 any one, it is characterized in that carry out in the following steps:

1) Strain activation: the strain of Trichoderma atroviride ( Trichoderma atroviride ) with the preservation number CGMCCNo.6828 was activated in potato dextrose agar medium for 72 hours and set aside;

The potato dextrose agar medium (abbreviated as PDA medium) consists of: 300 grams of potatoes, 20 grams of glucose, 15-20 grams of agar, 1000 milliliters of tap water, and natural pH;

2) Seed solution preparation: the activated Trichoderma dark green Tr1208 strain was cultured with wheat flour culture solution or Richard culture solution for 72-96 hours under shaking at 23-28°C, and the seed solution of Trichoderma dark green Tr1208 was obtained after cultivation;

The composition of the wheat flour culture solution is: 20g of whole wheat flour, 20g of glucose, 2g of yeast extract, 1000mL of water, pH 6.8-7.2;

The composition of Richard's culture medium is: potassium nitrate 10g, potassium dihydrogen phosphate 5g, magnesium sulfate 2.5g, ferric chloride 0.02g, sucrose 50g, water 1000mL, pH6.8～7.2;

3) Solid fermentation culture: add water 20~45% (w/w) to the solid fermentation medium material, the initial pH is between 6.5~7.5, high temperature sterilization, and then wait until the temperature drops below 30°C, insert the step 2) The seed liquid is then fermented and cultured for 96-148 hours at a temperature of 20-30°C, with an interval of 24-30 hours and regular stirring;

The composition of the solid fermentation medium (w/w) is: 2-4% wheat flour, 12-18% wheat bran, 77.1-84% edible mushroom waste residue or/and sawdust, 0.5-1% superphosphate , urea 0.2~0.5%, sucrose 0.2~0.5%, sterilized after moistening for use;

4) Measure the number of bacteria in the fermentation medium, and stop the fermentation culture after the spores in the fermentation medium reach more than 10 ⁹ cfu/g. The culture material is often dried at room temperature, pulverized by a universal pulverizer, and sieved to become dark green Trichoderma mother powder;

5) Preparation of Trichoderma dark green fungus: 2.0~60.0× ¹⁰⁸ spores/gram Trichoderma dark green Tr1208 mother powder, 0.05~0.1% xanthan gum, 1~1.5% sucrose, sodium humate or Potassium fulvic acid 0.05-0.2%, the rest is calcium carbonate and diatomite carrier, wherein the weight ratio of calcium carbonate:diatomite=1:2-5, mixed to prepare Trichoderma dark green fungus agent.

7. The application of the Tr1208 ( Trichoderma atroviride ) strain described in claim 1 in the preparation of fungal agents for the prevention and control of vegetable fungal diseases.

8. The dark green Trichoderma Tr1208 ( Trichoderma atroviride ) bacterial strain described in claim 1 is prepared as the control of leek gray mold, cucumber brown spot, cucumber powdery mildew, cucumber downy mildew, tomato early blight, melon and fruit blight , Fusarium wilt of melons and fruits, and application of fungal agents for root rot of melons and fruits.

9. The application of the Tr1208 ( Trichoderma atroviride ) bacterial strain described in claim 1 in the preparation of a bacterial agent for promoting plant growth.