CN101558766A - Trichoderma solid granules for preventing and controlling tobacco soil-borne fungus diseases and preparation method thereof - Google Patents
Trichoderma solid granules for preventing and controlling tobacco soil-borne fungus diseases and preparation method thereof Download PDFInfo
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Abstract
The invention discloses trichoderma solid granules for preventing and controlling tobacco soil-borne fungus diseases and a preparation method thereof, and aims to provide an effective, nontoxic, safe and residue-free bio-control microbial inoculum which is simple and convenient to use. The strain for producing the microbial inoculum is trichoderma harzianum TR13 with a preserving number of CGMCC No.2849. The preparation method comprises: (1) rejuvenescence of the strain; (2) preparation of the strain; (3) fermentation culture of a liquid; (4) fermentation culture of a solid; and (5) preparation.
Description
Technical field
The present invention relates to biopesticide, more particularly, the present invention relates to a kind of biologic product of preventing and treating tobacco soil-borne fungus diseases and preparation method thereof.
Background technology
Soil-borne disease of tobacco (soil borne tobacco diseases) is the comparatively serious big class disease of harm in the tobacco diseases, this class disease cause of disease can be survived in tobacco-growing soil, be bred, and infect root, root shank, or infect by root, root shank, cause root, root shank pathology, or the complete stool venereal disease becomes, its occurrence characteristic be popular for many years, increase the weight of year by year.The fungoid soil-borne disease takes place morely in these soil-borne diseases, endanger heavylier, and some becomes important economic disease in the tobacco agriculture production.This class fungal disease has black shank (Phytophthora parasitica var.nicotianae), damping off (Pythium spp.), damping off (Rhizoctonia solani) etc.Wherein, the heavier soil-borne disease that causes harm seedling stage is a damping off, and its cause of disease is mainly the mould pythium fungies such as (Pythium aphanidermatum) of melon and fruit corruption; The disease of field period outbalance has black shank.The cause of disease of these two kinds of diseases has highly adapted to cigarette strain root environment, causes the whole field period of cigarette strain all can be susceptible.General early susceptible, the disease loss is big more.Prior art adopts disease-resistant variety and wheel as the main anti-measure control of combining, but effect is unsatisfactory.
The biological control of the main silborne fungal diseases of tobacco has caused great attention both domestic and external.What current progress was the fastest is the biological control that is used for black shank, damping off, damping off with the biological prevention and control agent of wood mould (Trichoderma spp.) development.
Trichoderma is the desirable biocontrol microorganisms of a class, occupies important status on biocontrol of plant disease.At home and abroad the commodity biological prevention and control agent of registration has reached tens kinds at present.In recent ten years, the production technology of wooden mould biological prevention and control agent becomes better and approaching perfection day by day, and the microbial inoculum patented technology obviously rises.A little less than the blastopore life-span weak point and vitality that the employing liquid deep layer fermenting obtains; Solid culture can obtain relatively long conidium storage period, but the production cycle is long, the microbial inoculum spore content is low.Recent years, the maturation that the liquid-solid two-phase legal system is equipped with the high cryptogam technology of Trichoderma has strengthened the practicality process of Trichoderma greatly.But during liquid-solid two-phase method microbial inoculum was produced, the quality of solid culture was not easy control, has hindered the large-scale production of Trichoderma agent.Therefore, the industrialization of wooden removing mildew, standardization, commercialization production problem are still waiting further exploration.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art part, the trichoderma solid granules of a kind of effective, nontoxic, safety, noresidue, control tobacco soil-borne fungus diseases easy to use is provided.Simultaneously, the present invention also provides the preparation method of this trichoderma solid granules.
Purpose of the present invention is achieved by following technical proposals.
* except as otherwise noted, the percentage that is adopted among the present invention is mass percent.
The invention provides a kind of trichoderma solid granules of preventing and treating tobacco soil-borne fungus diseases.
Its classification called after Trichoderma harzianum (Trichoderma harzianum) of biocontrol bacterial strain of the present invention TR13, deposit number is CGMCC No.2849.
Production bacterial strain Trichoderma harzianum of the present invention (Trichoderma harzianum) TR13, separation is from Kunming, Yunnan rhizosphere soil, through cultivating proterties, morphology mensuration, this biocontrol fungi is a new bacterial strain TR13 of Trichoderma harzianum (Trichoderma harzianum).This bacterial strain has following feature: 1. bacterium colony growth rate on the PDA flat board becomes the concentric circles growth, fine hair shape, primary hyphae white, dirty-green when aging more than 1.5mm/h.2. optical microscope, usually 2～3 one group of sporophore, annular arrangement, the right angle stretches out, and is tree-shaped.Sporophore is short, and base portion attenuates, and expand the centre, long 3.0～4.3 μ m, middle wide 6.5～8.0 μ m, wide 2.0～3.2 μ m of base portion.Bottle stalk 2.0～2.8 μ m * 3.5～5.5 μ m, the conidium sphere, short obovate, base portion is truncate, spore size 2.5～3.5 μ m * 2.5～3.0 μ m.3. bacteriostasis is strong, can produce β-1,3 dextranases, cellulase, to balck shank (Phytophthoraparasitica var.nicotianae), damping off (Rhizoctonia solani), damping off (Pythiumspp., wherein based on Pythium aphanidermatum) etc. the tobacco soil-borne fungus cause of disease significant inhibitory effect is arranged, mainly show as hyperparasitism.4. colonization ability is strong, can promote the tobacco seedling growth decided at the higher level but not officially announced the growing of rhizosphere, root of tobacco.
The present invention is by the rejuvenation of spawn of Trichoderma harzianum (Trichoderma harzianum) TR13 bacterial strain, strain preparation, liquid fermentation and culture, solid fermentation is cultivated, the preparation trichoderma solid granules applies to it on tobacco, measures its control efficiency to the tobacco soil-borne fungus disease.
The invention provides a kind of preparation method of trichoderma solid granules, this method adopts following steps:
1. rejuvenation of spawn
Because trichoderma strain is a kind of biocontrol bacterial strain of facultative parasitism, the process of artificial culture is the similar saprophytic a kind of life style of nutrition often, and it is generally more to produce strain passage algebraically, and the parasitic ability of trichoderma strain is descended, and preparation weakens the prevention and control capability of disease.For keeping the preventive effect stability of preparation, must carry out rejuvenation to the bacterial classification of producing.
Concrete operations are as follows:
1) activation of bacterial classification
A spot of conidial powder on the picking TR13 bacterial classification, point are connected on the PDA flat board of new preparation, the no obvious condensed water in surface, 27.5 ± 1 ℃ of illumination cultivation 2～3 days.After waiting to grow bacterium colony, cut the mycelia piece (as far as possible unnecessary culture block being pruned) faster of growing, transfer again in the PDA flat board, 27.5 ± 1 ℃ of illumination cultivation 2～3 days, reject the flat board that pollutes, select that mycelial growth is dull and stereotyped faster to be continued to cultivate, the growth of regularly observing mycelia every day with produce the spore situation, select the starting strain of the big plate culture of fast growth, sporulation quantity as rejuvenation.
2) dull and stereotyped face-off is cultivated
The rejuvenation starting strain conidial powder point that picking is fresh is connected to a side of PDA flat board, fresh Rhizoctonia solani Kuhn (Rhizoctonia solani) (a kind of growth rate is the silborne fungal diseases cause of disease faster) the mycelia piece of opposite side inoculation Ф 4mm of distance 3～4cm.Postvaccinal dull and stereotyped 27.5 ± 1 ℃ of illumination cultivation 5～7 days are crossed the Rhizoctonia solani Kuhn mycelia until the mycelia of the starting strain of rejuvenation, and are produced till the spore thereon.
3) separation of bacterial classification, purifying
Rejuvenation starting strain during picking face-off is cultivated is at the spore that produces on the Rhizoctonia solani Kuhn bacterium colony rule on the PDA flat board separation, purifying, select the fast single bacterium colony of growth to transfer and carry out pure culture in the PDA flat board, in these pure cultures, select again and produce the culture that spore is fast, sporulation quantity is big, switching PDA inclined-plane, the bacterial classification behind the product spore is the bacterial classification of rejuvenation.
2. the preparation of bacterial classification
The quality of bacterial classification directly has influence on each operation of fermentation, and the requirement of quality and quantity is arranged.The requirement of matter comprises: 1. bacterial classification does not have living contaminants.Microexamination is that thalli morphology is normal, and body is consistent; 2. bacterial classification should be in identical physiological status, can grow fully and breed in the hope of bacterial classification, carries out synchronous growth.The slant strains of confession production usefulness is preserved in refrigerator and is no more than 7 days at most.The requirement of amount comprises: the 1. bacteria containing amount of bacterial classification.The seed liquor of bacterial classification contains the spore amount and is not less than 10
6Individual/ml.2. the inoculum concentration of bacterial classification.Need be definite by testing, consider to shorten fermentation period, control reproductive order of generation as far as possible.The volume of the seed liquor ratio with the volume of zymotic fluid is controlled in certain scope.
The preparation of bacterial classification at first activates 2 times from bacterial classification (as the sealing slant tube) the switching PDA flat board of preserving, and bottle is shaken in switching again, inoculates seeding tank then in certain proportion, and last culture transferring is to the fermenting and producing jar.Concrete steps are as follows:
(1) dull and stereotyped culture of strains
By sterile working from the dull and stereotyped bacterial classification of activation, the mycelia piece of picking 2～4mm * 2～4mm, 3～5 points are connected on the PDA flat board, put 27.5 ± 1 ℃, illumination cultivation 5～7 days.Note regular check every day flat board in the process of after inoculation, cultivating, should in time wash in a pan if any the culture of pollution or growth failure and remove.The quality standard of dull and stereotyped bacterial classification is: no living contaminants, and the product spore is fast, sporulation quantity is big, and color is dark green, and the spore size is consistent, and clear-cut is mixed with a small amount of mycelia.
(2) preparation of liquid spawn
From the dull and stereotyped bacterial classification of preparation, the mycelia piece of 6～8 2～4mm * 2～4mm of picking is added in the triangular flask (the liquid amount volumetric ratio is 30%～50%) that sterilization PD liquid is housed by sterile working, and 27.5 ± 1 ℃, illumination, 150r/m were cultivated 2 days.The standard of liquid spawn is: do not have assorted bacterium, thalline is in vigorous period, and mycelium is dense thick.
3. liquid fermentation and culture
Liquid spawn inserted with 2%～5% volume ratio fill liquid fermentation medium SDIS (prescription is: soy peptone 10g, sucrose 10g, dusty yeast 2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.3g, running water 1000ml, peanut oil 0.1%, defoamer 0.01%, pH nature.) fermentation tank in, 28 ± 1 ℃, with medium: air is 1: 1.6～2.2 volume ratio ventilation, and 120 rev/mins of stirrings of rotating speed were cultivated 36～42 hours.With liquid mycelia milky, thickness, mycelium dry weight 5mg/mL is above to be qualified zymotic fluid.
4. solid fermentation is cultivated
(1) inoculation
Qualified zymotic fluid is inoculated in solid fermentation medium RS[prescription by 20%～30% mass ratio is: rice husk 90%, the brown shorts 10% of 1～3mm, with mineral mineral salt (sulfate of ammoniac 2g, potassium dihydrogen phosphate 0.5g, sodium dihydrogen phosphate 0.5, magnesium sulfate 1g, calcium chloride 0.5g, the 1000ml running water) solution wetted, making its water content is 25%～30%.] in.During inoculation, stir with the zymotic fluid limit on the limit, to solid material water content 50%～60%.
(2) sabot
With the bottom stainless steel tray splendid attire of the screen cloth of 1～2mm, dark 4～6cm is housed, again tray is placed on the culturing rack.
(3) cultivate
Dark culturing: the material in the tray was cultivated 2～3 days under 28 ± 1 ℃, the dark condition of relative moisture 90%～95%, checked material, found that it is qualified that material inside and outside is covered with white hypha.
Illumination cultivation: the top at every layer of culturing rack adds light, illumination in 24 hours, under being 35 ± 1 ℃, the condition of relative moisture 50%～60%, ventilation by forced draft, temperature cultivated 2～3 days, turned over 1 time in per 12 hours, check that granular material is covered with green conidium, blood counting chamber count every gram dry material conidium content more than 109 for qualified.
5. preparationization
45 ± 1 ℃ of dryings of qualified solid culture 12～24 hours add the diatomite (protectant) of mass ratio 10%～15%, the Dexon powder (synergist) of mass ratio 0.1%～0.2%, and mixing is ground into the particle that particle diameter is 0.1～0.2mm, pack.
Compared with prior art, the present invention has following outstanding advantage:
1. adopt the liquid-solid two-phase one-step method of improvement to produce
Produce the bacterial strain mycelia that grows in liquid fermentation, produce spore in solid carrier, realize that the liquid-solid two-phase one-step method produces, overcome solid culture time shortcoming long, that pollute easily in the past, liquid-solid incubation time shortens 3～5 days.
Incubation is divided into 2 stages: the phase I is the liquid fermentation stage, the main bacterium slurry that is fit to solid culture of producing; Second stage is the solid culture stage, mainly is to make the surface of bacterium slurry attached to carrier, and produces spore under appropriate condition, and the nutrition of mycelial growth is mainly from the remaining nutrient component of liquid culture.
2. production procedure is workable, fidelity factor is high, success rate is high
During spawn culture, inoculum concentration is big, can make the bacterial classification of production capture matrix rapidly, forms the micropopulation of absolute predominance.During liquid culture, because the inoculum that this law adopts is the vigorous period mycelium that is in same physiological level, seeding tank can be realized synchronous growth, and its growth presents vigorous growth basically.During solid culture, mycelia does not utilize the nutrition in the solid material, pollution capable of blocking.
To the liquid-solid two-phase one-step method production procedure of designing, carried out test of many times, proved that the production procedure of liquid-solid two-phase one-step method is workable, fidelity factor is high, success rate high (can reach 100%), under the prerequisite of strengthening online detection, can stop fully to pollute, therefore, this flow process is practicable for the batch production production of Trichoderma solid particle agent.
3. liquid culture adopts the eutrophication medium
It is very fast in this medium (SDIS medium) growth to produce bacterial strain, can not form the disadvantageous hypertonic liquid of mycelia, and mycelia when being in vigorous period culture fluid also contain remaining nutrition, can satisfy mycelial growth on the solid carrier, produce the nutritional need of spore.
4. the solid culture medium permeability is strong
The primary raw material of solid culture medium is a husk, can strengthen the permeability of solid culture medium greatly, is beneficial to sterilization, also helps having cultivated the Trichoderma silk of gas, also help turn over, spore is produced in illumination.
5. solid culture is divided mycelial growth, is forced to produce 2 stages of spore
Dark culturing thermophilic, high humidity, dark help mycelia and adhere in solid matrix, grow; Illumination cultivation high temperature, low humidity, ventilation, printing opacity have been created good condition for the Trichoderma silk produces spore.
The explanation of preservation biomaterial
Its classification called after Trichoderma harzianum (Trichoderma harzianum) of biocontrol bacterial strain of the present invention TR13, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on 01 06th, 2009; The numbering CGMCC No.2849 that preservation is registered on the books.
Description of drawings
Fig. 1 is the schematic flow sheet that bacterial classification of the present invention is produced;
Fig. 2 is the process flow diagram of laboratory of the present invention lab scale;
Fig. 3 is the process flow diagram of workshop of the present invention pilot scale.
Embodiment
By specific embodiment given below and Application Example, can further be well understood to the present invention.But they are not the qualifications to protection domain of the present invention.
Embodiment 1
---the laboratory lab scale
Being laboratory fluids bacterium slurry preparatory phase from slant strains to shaking bottle bacterium slurry in the process chart, is workshop-based production process after the bacterium slurry inserts solid.Specific as follows:
1. dull and stereotyped culture of strains
By sterile working from the dull and stereotyped bacterial classification of activation, the mycelia of the big volume production spore of picking, 3～5 points are connected on the flat board, put 27.5 ± 1 ℃, illumination cultivation 5～7 days.Note regular check every day flat board in the process of after inoculation, cultivating, should in time wash in a pan if any the culture of pollution or growth failure and remove.For avoiding occurring owing to uneven illumination causes producing the inconsistent culture of spore, can reduce the stacking number of plies, as 2～3 layers of culture dish, if conditions permit can put one deck.
2. the preparation of liquid spawn
To burn red vaccinating lancet cooling, from the dull and stereotyped bacterial classification of preparation, the mycelia piece of 6～8 2～4mm * 2～4mm of picking is added in the 500ml triangular flask that 300ml sterilization PD liquid is housed by sterile working, and 27.5 ± 1 ℃ of illumination, 150r/m cultivated 2 days.Generally, shake bottle prepares with bacterial classification as far as possible on time in batches.
3. liquid fermentation and culture
Liquid spawn inserted with 2%～5% volume ratio fill liquid fermentation medium SDIS (prescription is: soy peptone 10g, sucrose 10g, dusty yeast 2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.3g, running water 1000ml, peanut oil 0.1%, defoamer 0.01%, pH nature.) triangular flask in, 28 ± 1 ℃, 150 rev/mins of shaken cultivation 36～42 hours.
4. solid fermentation is cultivated
4.1 inoculation
Qualified zymotic fluid is inoculated in solid fermentation medium RS[prescription by 20%～30% mass ratio is: rice husk 90%, the brown shorts 10% of 1～3mm, with mineral mineral salt (sulfate of ammoniac 2g, potassium dihydrogen phosphate 0.5g, sodium dihydrogen phosphate 0.5, magnesium sulfate 1g, calcium chloride 0.5g, the 1000ml running water) solution wetted, making its water content is 25%～30%.] in.During inoculation, manually stir with the zymotic fluid limit on the limit, grab material to hand and firmly pinch, finger stitch firm water outlet ooze till (water content 50%～60%).
4.2 sabot
With the bottom stainless steel tray splendid attire of the screen cloth of 1～2mm, dark 4～6cm is housed, again tray is placed on the culturing rack.
4.3 cultivate
Dark culturing: the material in the tray was cultivated 2～3 days under 28 ± 1 ℃, the dark condition of relative moisture 90%～95%.For keeping humidity, avoid cross pollution, the plastic seal culturing rack of available sterilization.
Illumination cultivation: the top at every layer of culturing rack adds light, carry additionally the 40W fluorescent tube, illumination in 24 hours is to cultivate 2～3 days under the condition of 35 ± 1 ℃, relative moisture 50%～60% (regulating the ventilating opening size realizes), ventilation by forced draft in temperature, turns over 1 time in per 12 hours.
5. preparationization
45 ± 1 ℃ of constant temperature oven dryings of qualified solid culture 12～24 hours; add the diatomite (protectant) of mass ratio 10%～15%, the Dexon powder (synergist) of mass ratio 0.1%～0.2%, mixing is ground into the particle that particle diameter is 0.1～0.2mm; quality inspection, pack.Promptly get solid particle agent of the present invention.
Embodiment 2
---the workshop pilot scale
The workshop pilot process improves on lab scale basis, laboratory and forms, and its processing step is as follows:
1. dull and stereotyped culture of strains, 2. the preparation of liquid spawn is with embodiment 1.
3. liquid fermentation and culture
Liquid spawn inserted with 2%～5% volume ratio fill in the fermentation tank of liquid fermentation medium SDIS, 28 ± 1 ℃, with medium: air is 1: 1.6～2.2 volume ratio ventilation, and 120 rev/mins of stirrings of rotating speed were cultivated 36～42 hours.
4. solid fermentation is cultivated
Inoculation: qualified zymotic fluid is inoculated among the solid fermentation medium RS by 20%～30% mass ratio.During inoculation, with through the agitator of steam sterilizing, stir with the zymotic fluid limit on the limit, grab material to hand and firmly pinch, finger stitch firm water outlet ooze till (water content 50%～60%).
Sabot: with the bottom stainless steel tray splendid attire of the screen cloth of 1～2mm, dark 4～6cm is housed, again tray is placed on the culturing rack.
Dark culturing: the material in the tray was cultivated 2～3 days under 28 ± 1 ℃, the dark condition of relative moisture 90%～95%.For keeping humidity, can use humidifier.
Illumination cultivation: the top at every layer of culturing rack adds light, carry additionally the 40W fluorescent tube, illumination in 24 hours is to cultivate 2～3 days under the condition of 35 ± 1 ℃, relative moisture 50%～60% (humidifier, dehumidifier, forced ventilation etc. are common to be realized), ventilation by forced draft in temperature, turns over 1 time in per 12 hours.
5. preparationization
45 ± 1 ℃ of vibra fluidized bed dryings of qualified solid culture 4～6 hours; add the diatomite (protectant) of mass ratio 10%～15%, the Dexon powder (synergist) of mass ratio 0.1%～0.2%, mixing is ground into the particle that particle diameter is 0.1～0.2mm; quality inspection, pack.Promptly get solid particle agent of the present invention.
Application Example 1
---prevent and treat the effect test of tobacco damping off seedling stage
1. materials and methods
1.1 material
Test medicine: solid particle agent of the present invention, 50% carbendazim.
Test tobacco bred: K326
Test site: Yunnan Provine Tobacco Science Inst. plastic greenhouse floating seedlings pond.
1.2 test method
Seed pelleting: the ratio in coating agent for seed 1% adds solid particle agent of the present invention, 50% carbendazim respectively, naked kind to tobacco bred K326 is carried out dressing, make blank not add the capsuled seed of preventing and treating the damping off medicament, form 3 types capsuled seed altogether.
The preparation of seedling medium in spite of illness: melon and fruit corruption mould (main pathogen of tobacco damping off) was cultivated 10～15 days for 28 ± 1 ℃ with the wheat bran solid culture medium of 1～3mm, pour out, smash to pieces, naturally dry, the seedling medium mixing of mass ratio with 2% and sterilization can prepare the seedling medium of being with da mping-off fungi.
Test is handled: the seedling medium that will prepare the band da mping-off fungi floating seedlings dish of packing into, sow 3 types capsuled seed more respectively, and every type of capsuled seed of sowing forms 1 test and handles totally 3 processing.Every processing sowing 1 dish (about 280 capsuled seeds), 3 repetitions, dish is in same nursery pond randomized arrangement.
State of an illness investigation: sowing finishes, and grows seedlings by normal floating seedlings measure, to the investigation of grand cross phase, mainly investigates the incidence of disease, calculates relative control efficiency.
2. result and analysis
Prevent and treat tobacco damping off test effect seedling stage and see Table 1, can find out that from table 1 microbial inoculum of the present invention reaches 75.36% to the control efficiency of tobacco damping off, omit control efficiency, but differ not obvious inferior to 50% carbendazim 78.58%.Therefore, solid particle agent of the present invention can be used as the control medicament use of tobacco damping off.
Table 1. is prevented and treated tobacco damping off test effect seedling stage
Application Example 2
---the effect test of field period control black shank
1. materials and methods
1.1 material
Test medicine: solid particle agent of the present invention, 48% metalaxyl-mn-zn.
Test tobacco bred: the big gold dollar of safflower
Test site: Yunnan Provine Tobacco Science Inst. grinds and the proving ground.
1.2 test method
The preparation of organic medicine fertilizer: solid particle agent of the present invention and the fertilizer (not containing made chemical fertilizer) that becomes thoroughly decomposed are with 1: 10 ratio mixing of quality, 48% metalaxyl-mn-zn and the ratio mixing of the fertilizer that becomes thoroughly decomposed equally with 1g/kg form 2 kinds of organic medicine fertilizer respectively.
The preparation of black shank bacterium paddy: identify that by the tobacco bred disease resistance method described in the YC/T 41-1996 industry standard prepares black shank bacterium paddy.
Test is handled: 2 kinds of organic medicine fertilizer that will prepare, when normal root water is watered in the cigarette transplantation of seedlings, by the 40g/ strain impose on transplant the cigarette seedling around, the training moisture in the soil of loosening the soil.Make blank with the fertilizer that becomes thoroughly decomposed equally, form 3 processing altogether, cigarette 50～80 strains are planted in every processing.Seeding row spacing setting, fertilising, management are produced popularization with local tobacco and are required identical.Each handles the sub-district randomized arrangement that forms, 3 repetitions.
Inoculation bacterium paddy: organic medicine fertilizer was used back 20 days, pressed the tobacco bred disease resistance and identified the method inoculation black shank bacterium paddy described in the YC/T 41-1996 industry standard.After the inoculation, irritate semlsulcus water and preserved moisture 2～3 days.
State of an illness investigation: inoculation black shank bacterium paddy same day, the industry standard YC/T39-1996 by the tobacco diseases investigation carried out a state of an illness radix investigation, reinvestigated the state of an illness one time, and calculated disease index, preventive effect in 40 days.
2. result and analysis
Field period control black shank test effect sees Table 2, can find out that from table 2 microbial inoculum of the present invention reaches 78.91% to the control efficiency of black shank, omits the control efficiency inferior to 48% metalaxyl-mn-zn 82.50%, but differs not obvious.Therefore, solid particle agent of the present invention can be used as the control medicament use of black shank.
Table 2. field period control black shank test effect
Claims (8)
1. trichoderma solid granules of preventing and treating tobacco soil-borne fungus diseases, it is characterized in that: the production bacterial strain of this microbial inoculum is Trichoderma harzianum (Trichoderma harzianum) TR13, preserving number CGMCC No.2849.
2. trichoderma solid granules according to claim 1, it is characterized in that: the culture medium prescription of (1) described bacterial strain is as follows: 1. test tube slant medium PDA prescription is: fresh potato 200g (liquor, cross the elimination residue, reserved filtrate), sucrose 20g, agar 17～20g, distilled water 1000ml, pH nature; 2. liquid fermentation medium SDIS prescription is: soy peptone 10g, sucrose 10g, dusty yeast 2g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.3g, running water 1000ml, peanut oil 0.1%, defoamer 0.01%, pH nature; 3. solid fermentation medium RS prescription is: the brown shorts 10% of rice husk 90%, 1～3mm, with mineral mineral salt (sulfate of ammoniac 2g, potassium dihydrogen phosphate 0.5g, sodium dihydrogen phosphate 0.5, magnesium sulfate 1g, calcium chloride 0.5g, the 1000ml running water) solution wetted, making its water content is 25%～30%; (2) its microbial inoculum production adopts the liquid-solid two-phase one-step method to carry out, and promptly produces on the liquid on mycelia, the carrier and produces spore.
3. the preparation method of trichoderma solid granules according to claim 1 and 2 is characterized in that may further comprise the steps:
(1) rejuvenation of spawn; (2) preparation of bacterial classification; (3) liquid fermentation and culture; (4) solid fermentation is cultivated; (5) preparationization.
4. the preparation method of trichoderma solid granules according to claim 3 is characterized in that, described rejuvenation of spawn is to adopt following steps:
(1) activation of bacterial classification
A spot of conidial powder on the picking TR13 bacterial classification, point are connected on the PDA flat board of new preparation, the no obvious condensed water in surface, 27.5 ± 1 ℃ of illumination cultivation 2～3 days; After waiting to grow bacterium colony, cut the mycelia piece (as far as possible unnecessary culture block being pruned) faster of growing, transfer again in the PDA flat board, 27.5 ± 1 ℃ of illumination cultivation 2～3 days, reject the flat board that pollutes, select that mycelial growth is dull and stereotyped faster to be continued to cultivate, the growth of regularly observing mycelia every day with produce the spore situation, select the starting strain of the big plate culture of fast growth, sporulation quantity as rejuvenation;
(2) dull and stereotyped face-off is cultivated
The rejuvenation starting strain conidial powder point that picking is fresh is connected to a side of PDA flat board, the fresh Rhizoctonia solani Kuhn mycelia piece of opposite side inoculation Φ 4mm of distance 3～4cm; Postvaccinal dull and stereotyped 27.5 ± 1 ℃ of illumination cultivation 5～7 days are crossed the mycelia of Rhizoctonia solani Kuhn until the mycelia of the starting strain of rejuvenation, and are produced till the spore thereon;
(3) separation of bacterial classification, purifying
Rejuvenation starting strain during picking face-off is cultivated is at the spore that produces on the Rhizoctonia solani Kuhn bacterium colony rule on the PDA flat board separation, purifying, select the fast single bacterium colony of growth to transfer and carry out pure culture in the PDA flat board, in these pure cultures, select again and produce the culture that spore is fast, sporulation quantity is big, switching PDA inclined-plane, the bacterial classification behind the product spore is the bacterial classification of rejuvenation.
5. the preparation method of trichoderma solid granules according to claim 3 is characterized in that,
The preparation of described bacterial classification is to adopt following steps:
(1) dull and stereotyped culture of strains
By sterile working from the dull and stereotyped bacterial classification of activation, the mycelia piece of picking 2～4mm * 2～4mm, 3～5 points are connected on the flat board, put 27.5 ± 1 ℃, illumination cultivation 5～7 days.Note regular check every day flat board in the process of after inoculation, cultivating, should in time wash in a pan if any the culture of pollution or growth failure and remove; The quality standard of dull and stereotyped bacterial classification is: no living contaminants, and the product spore is fast, sporulation quantity is big, and color is dark green, and the spore size is consistent, and clear-cut is mixed with a small amount of mycelia;
(2) preparation of liquid spawn
From the dull and stereotyped bacterial classification of preparation, the mycelia piece of 6～8 2～4mm * 2～4mm of picking is added in the triangular flask that sterilization PD liquid is housed by sterile working, and the liquid amount volumetric ratio is that 30%～50%, 27.5 ± 1 ℃, illumination, 150r/m were cultivated 2 days; The standard of liquid spawn is: do not have assorted bacterium, thalline is in vigorous period, and mycelium is dense thick.
6. the preparation method of trichoderma solid granules according to claim 3 is characterized in that, described liquid fermentation and culture is to adopt following steps:
(1) liquid spawn is inserted with 2%～5% volume ratio filled in the fermentation tank of liquid fermentation medium SDIS; (2) under 28 ± 1 ℃, with medium: air is 1: 1.6～2.2 volume ratio ventilation, and 120 rev/mins of stirrings of rotating speed were cultivated 36～42 hours; With liquid mycelia milky, thickness, mycelium dry weight 5mg/mL is above to be qualified zymotic fluid.
7. the preparation method of trichoderma solid granules according to claim 3 is characterized in that, it is to adopt following steps that described solid fermentation is cultivated:
(1) inoculation
Qualified zymotic fluid is inoculated in solid fermentation medium RS[prescription by 20%～30% mass ratio is: rice husk 90%, the brown shorts 10% of 1～3mm, with mineral mineral salt (sulfate of ammoniac 2g, potassium dihydrogen phosphate 0.5g, sodium dihydrogen phosphate 0.5, magnesium sulfate 1g, calcium chloride 0.5g, the 1000ml running water) solution wetted, making its water content is 25%～30%.] in.During inoculation, stir with the zymotic fluid limit on the limit, to solid material water content 50%～60%;
(2) sabot
With the bottom stainless steel tray splendid attire of the screen cloth of 1～2mm, dark 4～6cm is housed, again tray is placed on the culturing rack;
(3) cultivate
Dark culturing: the material in the tray was cultivated 2～3 days under 28 ± 1 ℃, the dark condition of relative moisture 90%～95%, checked material, found that it is qualified that material inside and outside is covered with white hypha;
Illumination cultivation: the top at every layer of culturing rack adds light, illumination in 24 hours, under being 35 ± 1 ℃, the condition of relative moisture 50%～60%, ventilation by forced draft, temperature cultivated 2～3 days, turned over 1 time in per 12 hours, check that granular material is covered with green conidium, blood counting chamber is counted every gram dry material conidium content 10
9More than individual is qualified.
8. the preparation method of trichoderma solid granules according to claim 3, it is characterized in that, described preparationization is to adopt following steps: 45 ± 1 ℃ of dryings of (1) qualified solid culture 12～24 hours, and the diatomite that adds mass ratio 10%～15% is made protectant; The Dexon powder of mass ratio 0.1%～0.2% is made synergist; (2) mixing is ground into the particle that particle diameter is 0.1～0.2mm, pack.
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| CN102524306A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Preparation method of trichoderma |
| CN103451111A (en) * | 2013-09-02 | 2013-12-18 | 中国农业科学院农业资源与农业区划研究所 | Saline-alkali tolerant trichoderma harzianum and application thereof |
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| CN1337164A (en) * | 2000-08-09 | 2002-02-27 | 玉溪红塔烟草(集团)有限责任公司 | Microbial bactericide and its prepn. |
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| CN102524306A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Preparation method of trichoderma |
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| CN108911863A (en) * | 2018-08-16 | 2018-11-30 | 河南科技大学 | Tobacco field probiotics and preparation method thereof with prevention and control soil-borne disease function |
| CN109337829A (en) * | 2018-12-18 | 2019-02-15 | 山东五福生生态工程有限公司 | Trichoderma harzianum solid fermentation method, solid fermentation product, Trichoderma harzianum microbial inoculum and its preparation method and application |
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