CN1786150A - Green tricoderma LTR-2 strain and its preparation - Google Patents
Green tricoderma LTR-2 strain and its preparation Download PDFInfo
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Abstract
The invention relates to viridin LTR-2 strain and its preparation. It belongs to biotechnology field. The preserving number of Trichoderma viride strain LTR-2 is CGMCC NO.1498. It is scalped from vegetable field plant root soil series earth. Its features are that colony grows quickly on PDA flat; hypha layer is thicker; compact clump is bunchiness; it is white at initial stage, then it turns to bottle green; spore production area is always arranged concentric rotate. It has higher enzyme activity, and is used to make plant antibiotics and biological control preparation.
Description
Technical field
The present invention relates to viride (Trichoderma viride) LTR-2 bacterial strain, its screening method and relevant preparation and preparation method, belong to biological technical field.
Background technology
Trichoderma (Trichoderma spp.) belongs to the hyphomycetes of imperfect fungi, Moniliales, Moniliaceae, be present in extensively that soil, rhizosphere, leaf enclose, in the ecotope such as seed and bulb, because extensive adaptability, broad spectrum and the many mechanisms of Trichoderma are the key objects of disease flocking biocontrol scholar research always.
Wei Lin, the lignin Trichoderma is to the strong parasitization of dry thread Pyrenomycetes and other soil-borne fungus, Plant Pathology, 1932,22:837-845 (WEILDING R.Studies on a lethal principle effective in the parasiticaction of Trichoderma hamatum on Rhizoctonia solani and other soil fungi, Phytopathology, 1932,22:837-845.) find that the lignin Trichoderma can parasitize many soil-borne fungus under culture condition, increase its content and can prevent and treat some pathomycete in soil, Trichoderma causes people's attention thus.Have now found that trichoderma harzianum, trichoderma viride, koning trichoderma bacterium, hook-shaped Trichoderma and long shoot Trichoderma etc. show antagonistic activity to the various plants pathogenic bacteria.Like to draw the Supreme Being, the bavin tower, Boyle etc., by the parasitization of electron microscope and opticmicroscope scanning Trichoderma to dry thread Pyrenomycetes and neat pyrenomycetes, Plant Pathology, 1983,73:85-88 (ELAD Y, CHET I, BOYLEP, et al.Parasitism of Trichoderma spp.on Rhizoctonia solani and Sclerotium rolfsiiscanning electron microscopy and fiuorescence microscopy, Phytopathology, 1983,73:85-88.) studies show that, after Trichoderma and the identification of host fungi, the Trichoderma silk twines growth along the parallel growth of host mycelia and spirrillum, and produces appressorium shape branch and be adsorbed on the host mycelia, by secretion extracellular enzyme dissolved cell wall, penetrate the host mycelia, draw nutrition.Most scholars think that the antagonism Trichoderma has produced the lytic enzyme of a series of degraded pathogenic bacteria cell wallss in parasitic processes, and the extracellular enzyme relevant with the biological and ecological methods to prevent plant disease, pests, and erosion effect of antagonism Trichoderma mainly is chitinase and beta-1,3-glucanase.Trichoderma has advantage aspect the generation antimicrobial substance, and with regard to its kind, only antimycotic meta-bolites is at least more than 70 kinds.The Horace, the Richard, Nice of ancient India etc., breathe out the 6-pentyl pyrone of wooden mould generation now: plant growth inhibitor and sterilant, agricultural biochemistry, 1986,50 (11): 2943-2945 (HORACE G C, RICHARD H C, FANIST G, et al.6-Pentyl-Q-pyrone from Trichoderma harzianum:Its plant growth inhibitory andantimicrobial properties.Agric Biol Chem, 1986,50 (11): 2943-2945.) report, one of main mechanism of trichoderma harzianum control dry thread Pyrenomycetes is exactly to have produced the volatility microbiotic, through being accredited as 6-pentyl pyrone.It is comprehensive that the antagonistic action of Trichoderma is considered to that 2 kinds or 3 kinds of mechanism act on simultaneously or sequentially.The Bake, in husband gentry this, handle the biological control device that pea seed reduces disease with Trichoderma, Plant Pathology, 1986,76:720-725 (BAKERR, LIFISHITZ R, Mechanism of biological control of precmergence damping-off of peaby sead treatment with Trichoderma spp.Phytopathology,) studies show that, behind Trichoderma processing pea seed, Trichoderma comprises microbiotic and the two kinds of mechanism of hyperparasitism of producing to the mould effect of corruption.
Along with the day by day care of the public to environment and health problem, the research of biological control has been subjected to widely with application and has paid attention to, in the antagonistic microbe of having found at present, especially in plant soil-borne disease fungal pathogens biological control work, often the Trichoderma of report is the most promising biological and ecological methods to prevent plant disease, pests, and erosion factor, as a kind of important biocontrol of plant disease bacterium, the knowledge in this field has been enriched in the research of Trichoderma, has increased the understanding of people to biocontrol of plant disease.The commercialization of multiple Trichoderma preparation will encourage the associating of research institution and enterprise, thereby research of Trichoderma and application are developed to more deep direction widely.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of viride (Trichoderma viride) bacterial strain LTR-2 is provided, and screening method.
The present invention also provides the purposes of viride bacterial strain LTR-2, i.e. viride bacterial strain LTR-2 Agrotechnical formulation.
1, green tricoderma LTR-2-2, preserving number are CGMCC NO.1498.Green tricoderma LTR-2-2 strain characteristics is described below:
Bacterium colony is grown on the PDA flat board soon, and subiculum is thicker, fine and close clump pencil, and the initial stage is white, and is smooth, the later stage is deep green because of producing conidium.Produce the spore district and often be arranged in the concentric wheel stripe shape.The bacterium colony back side is colourless, is light yellow sometimes.Mycelia is transparent, have every, cell walls is smooth, conidiophore is uprightly born by mycelia, and is colourless, branch is many, to giving birth to or two to three grades of branches of alternate, integral body resembles branch; Branch and conidiophore approximate right angle, end is a stigma, the stigma doleiform, conidium sphere or oblong, surface irregularity perhaps is covered with little furcella; Unit cell leans on mucus to be gathered into the conidial head of spherical green on stigma.The result as depicted in figs. 1 and 2.
2, the screening method of green tricoderma LTR-2-2 is as follows:
(1) sample collecting: collected specimens is a vegetables field soil, and the place is Jinan, pushes top layer earth aside with spades, until seeing the soil layer that root system of plant exists, the root of soil together with plant is shoveled out, installs with polyethylene bag, takes back the laboratory.
(2) separation method: root system is air-dry slightly together with soil, pat root system, adhesion soil is come off, then with residual soil on the abundant rinsing root of aqua sterilisa, keep rinsing liquid, make gradient dilution, and get 0.1mL respectively and splash on the TSM culture medium flat plate with stroke-physiological saline solution, L shaped glass stick with sterilization smoothens, and puts in 28 ℃ of thermostat containers and cultivates 2-4 days.Single bacterium colony of the approximate Trichoderma of picking colony form, the dull and stereotyped purifying of switching PDA is cultivated, and is Trichoderma through the microscopy preliminary evaluation, and numbering is stored on the PDA inclined-plane.
Above-mentioned PDA substratum: the beans 200g that fetches earth, clean and to be cut into small pieces, use water boil 30min, use filtered through gauze, keep filtrate, add 10g glucose, 15g agar, be settled to 1000mL, 121 ℃ of sterilization 20min are standby, and aseptic technique adds paraxin makes the substratum chloromycetin content reach 100 μ g/mL.
Above-mentioned TSM substratum (the half selected substratum of selecting of Trichoderma): MgSO
47H
2O 0.2g, K
2HPO
40.9g, KCl 0.15g, NH
4NO
31.0g, glucose 3.0g, rose-red 0.15g, 60% fenaminosulf wettable powder 0.3g, PCNB 0.2g, agar 15g, water 1000mL.121 ℃ of sterilization 20min are standby, and aseptic technique adds paraxin makes the substratum chloromycetin content reach 100 μ g/mL.
3, the solid fermentation of green tricoderma LTR-2-2 is cultivated, and carries out according to the following steps, below is weight ratio or weight percent.
(1) slant strains: adopt solid PDA substratum, this green tricoderma LTR-2-2 is seeded on the test tube substratum, cultivated 2-3 days for 28 ℃.
(2) eggplant bottle bacterial classification: adopt liquid PDA substratum, test tube strains be seeded in the liquid eggplant bottle, place on the shaking table 28 ℃ shaking culture 3-4 days.
(3) liquid spawn: adopt seed culture medium, Semen Maydis powder 2%, glucose 0.5%, soybean cake powder 1%, dipotassium hydrogen phosphate 0.2%, potassium primary phosphate 0.3%, lime carbonate 1%, pH6.0 sterilized 40 minutes for 121 ℃, the seed of 1-2 eggplant bottle is washed with sterilized water, be inoculated in the seeding tank of 150 liters 30 ℃ of cultivations, air flow 1: 0.6-0.8, stirring velocity is 200r/min, and incubation time is 36-48 hour.
(4) solid spawn: adopt solid medium, wheat bran: rice husk: Semen Maydis powder=7: 1: 2, water content is 70% (comprising the moisture of the liquid spawn that inoculates), pH6.0,121 ℃ of sterilizations were cooled off with recirculated water after 40 minutes, in the combined inoculation device liquid spawn and solid medium are mixed, inoculum size is 5-10%, and inoculation finishes and transfers to the solid culture indoor cultivation.
(5) cultivate: substratum thickness is 5cm, and the material temperature control is at 30 ± 2 ℃, and room temperature is controlled at 25-30 ± 2 ℃, and the relative water content of air is controlled at 95-100%, and incubation time is 5-7 days.
Cultivation finishes, and with the solid culture natural air drying, the finished product water content is controlled at 5-10%, gets green tricoderma LTR-2-2 solid culture.Cross 100 mesh sieves and collect spore powder.This spore powder can be used for preparing Agrotechnical formulation.
4, the biological activity determination method of green tricoderma LTR-2-2 is as follows:
(1) bacteriostasis of green tricoderma LTR-2-2 is measured: move into the pathogenic bacteria bacterium cake of diameter 5mm respectively in the dull and stereotyped central authorities of PDA, survey bacterial strain in the reception of distance bacterium cake 2.5cm place's point, cultivated 5-7 days for 28 ℃, write down antibacterial bandwidth, 3 repetitions are established in each processing.
Indication pathogenic strains: eliminating vegetable botrytis bacterium (Botrytis cinerea) Bc-6, fusarium moniliforme (Fusariummoniliforme) Fm26, wheat root-rot sickle-like bacteria (Fusarium graminearum) Fg2, long spore bacterium (Helminthosporium sativum) Hsll of wriggling of wheat, ring rot of apple bacterium (Macrophoma kawatsukai) Mk8, cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum) V19, this research department separates preservation; Sheath blight fungus (Rhizoctoniacerealis) RC46 and gaeumannomyces graminis (Gaeumannomyces graminisvar.tritici) V32 is provided by China Agricultural University.The antimicrobial spectrum of green tricoderma LTR-2-2 sees Table 1, and the dull and stereotyped restraining effect of green tricoderma LTR-2-2 pair cotton-wilt fusarium is seen Fig. 3.
The antimicrobial spectrum of table 1 green tricoderma LTR-2-2 (mm)
Annotate: data are three multiple mean values.
(2) the short living ability of green tricoderma LTR-2-2
Cucumber seeds (capital new 2000) green tricoderma LTR-2-2 spore suspension (10
8Cfu/mL) be seeded in the healthy soil behind the immersion 30min, 20 in every basin, triplicate, with clear water in contrast.(15-30 ℃) cultivates long, the total dry weight of investigation individual plant main root of all sampling after 45 days in the greenhouse.The results are shown in Table 2.
Stimulation ratio %=(handling total dry weight-contrast total dry weight) * 100%/contrast total dry weight
Short the giving birth to of table 2 green tricoderma LTR-2-2 tested
Annotate: data are three multiple mean values.
(3) the greenhouse biocontrol effect experiment of green tricoderma LTR-2-2
Cotton seeds (cotton No. 12 of Shandong) is used green tricoderma LTR-2-2 spore suspension (10
8Cfu/mL) be seeded into behind the immersion 60min in the rhizoctonia rot of cotton morbidity soil, 20 in every basin, triplicate, with clear water in contrast.(15-30 ℃) cultivates after 42 days all sampling investigation biomass of individual tree, cotton seedling blight incidences in the greenhouse, calculates each bacterial strain to the prevention effect of cotton seedling blight and to the influence of cotton total dry weight.The results are shown in Table 3.
Cotton seedling blight state of an illness investigation grade scale:
0: anosis; 1: basal part of stem has little scab, accounts for below 1/4 of stem girth; 2: the basal part of stem scab is bigger, accounts for the 1/4-1/2 of stem girth; 3: the basal part of stem scab accounts for more than 1/2 of stem girth, but does not destroy whole stem girth as yet; 4: the basal part of stem scab occupies whole stem girths, plant death.
Total strain number of diseased plant number * 100%/each processing of each processing of diseased plant rate %=
Disease index %=∑ (each sick level * strain numbers at different levels) * 100%/(the highest sick grade * always investigate strain number)
Preventive effect %=(contrast disease index-processing disease index) * 100%/contrast disease index
Stimulation ratio %=(handling total dry weight-contrast total dry weight) * 100%/contrast total dry weight
Table 3 green tricoderma LTR-2-2 pair cotton seedling blight prevention effect and greenhouse volume increase test
Annotate: data are three multiple mean values.
(4) the lyase determination of activity of green tricoderma LTR-2-2
Beta-1,3-glucanase determination of activity: preparation dextran substratum, KH
2PO
46.8g, K
2HPO
417.98g, yeast water 5g, Poria powder 4g, aniline blue 100mg, agar powder 15g, water 1000mL.Connect green tricoderma LTR-2-2 bacterium sheet (diameter is 5mm) in dull and stereotyped central point, cultivated the size of observing colourless hydrolysis area 2-4 days for 28 ℃.
Chitinase determination of activity: preparation chitin substratum, with acetone that the 0.2kg chitin is moistening, place 1000mL HCl, ice bath spends the night, when chitin is separated into small-particle, suspension is placed distilled water, chitin is settled out, and abandons supernatant liquor, washes repeatedly with distilled water, up to chitin suspension is neutral, gets 1000mL soliquid (including the 3-5g chitin) and adds following substances: (NH
4)
2SO
43g, KH
2PO
44g, K
2HPO
43g, MgSO
47H
2O 0.2g, yeast water 0.2g, H
3BO
45.6mg, CuSO
45H
2O 0.4mg, ZnSO
4H
2O 0.5mg, NaMoO
42H
2O 1.5mg, ironic citrate 1mg, CaCl
210mg, agar 10-15g, pH7.2-7.4.Connect green tricoderma LTR-2-2 bacterium sheet (diameter is 5mm) in dull and stereotyped central point, cultivated the size of observing transparent hydrolysis area 2-4 days for 28 ℃.
Inscribe determining enzymic activity of beta-glucan: prepare 1% carboxymethyl cellulose substratum, 1% carboxymethyl cellulose, 0.87% dipotassium hydrogen phosphate, 0.252% citric acid, 1.5% agar.Connect green tricoderma LTR-2-2 bacterium sheet (Φ 5mm) in dull and stereotyped central point, cultivated 3 days for 28 ℃, cultivate and finish, with the dull and stereotyped 20min of 1mg/mL congo red staining, then with the 1mol/L NaCl 20min that fades, and clean with flushing with clean water, observe having or not and size of transparent circle under fluorescent light.The results are shown in Table 4.
The β of table 4 green tricoderma LTR-2-2-1,3 dextranase activity, chitinase activity, inscribe 1,4 beta-glucanase activity
| Dextranase activity | The chitinase activity | ||||||||||
| Colony diameter (cm) | Hydrolysis loop diameter (cm) | Colony diameter (cm) | Hydrolysis loop diameter (cm) | ||||||||
| 48hr | 72hr | 48hr | 72hr | 48hr | 72hr | 48hr | 72hr | ||||
| 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | ||||
| 2.0 | 2.5 | 3.6 | 3.8 | 1.6 | 2.4 | 3.2 | 4.0 | 3.6 | 8.4 | 2.0 | 4.2 |
Annotate: 1: β-1,3 dextranase activity, 2: the inscribe 1,4 beta-glucanase activity.
Trichoderma LTR-2 enzyme system is more complete, and has higher enzymic activity, and this also is that it has one of reason of high antagonism.
(5) microbiotic pyrone (2H-Pyran-2-one, 5, preparation 6-dihydro-6-pentyl-): cultivate green tricoderma LTR-2-2 with the solid bran mass, obtain spore powder.Soaked spore powder 2 days with methylene dichloride 5-10 ℃, soak solution is successively through 5% charcoal absorption, the washing of 3% sodium carbonate solution, anhydrous sodium sulfate dehydration, at last treatment solution is evaporated to thickly, obtains containing the enriched material of microbiotic pyrone, the molecular structure of this microbiotic pyrone is
The product pyrone of above-mentioned steps is to the dull and stereotyped restraining effect (see figure 4) of the PDA of dry thread Pyrenomycetes: 1 is contrast, 2 is pyrone, on the dull and stereotyped vertical crossed lines of PDA, beat the hole of getting 4 diameter 5mm apart from central 35mm place, the concentration that splashes into 50uL in 2 holes (indicating 2 among Fig. 3) is the pyrone of 200 μ g/mL (solvent is a methylene dichloride), splash into control solvent methylene dichloride in addition in 2 holes (indicating 1 among Fig. 3) with volume, dull and stereotyped central authorities insert the dry thread Pyrenomycetes bacterium sheet of diameter 5mm, cultivate the fungistatic effect of observing pyrone in 2-3 days for 25 ℃.Pyrone has had strong inhibitory effects to dry thread Pyrenomycetes as a result, and antibacterial band size is 6.5mm (minimum value between hole preglabellar field and pathogenic bacteria bacterium colony outward flange), and the contrast hole has covered with pathogenic bacteria.
(6) the hyperparasite ability of green tricoderma LTR-2-2
Dry thread Pyrenomycetes and Trichoderma LTR-2 are cultivated in face-off on the PDA flat board, after the contact of two bacterium colonies, directly place microscopically to observe the supercrescence of antagonist flat board to dry thread Pyrenomycetes, comprise the parasitic point of antimicrobial short of money on the unit dry thread Pyrenomycetes mycelia, the parasitic mode of mycelia, be in microscopically counting dry thread Pyrenomycetes mycelia by the percentage ratio of Trichoderma parasitism two bacterium colonies intersections, the power of expression parasitic ability.
In the face-off experiment, the bacterium colony of observing Trichoderma and dry thread Pyrenomycetes about about the 4 days mould germ bacterium colonies that cover fully of wood in back that intersect, and volume production spore greatly show green; Mycelia parasitic rate to each dry thread Pyrenomycetes reaches 100%.Can see that at microscopically Trichoderma has tangible supercrescence to dry thread Pyrenomycetes, parasitic mode comprises winding, parallel growth, penetrates etc.Observation shows, particularly the aged mycelia parasitic ability of wilt is not strong to the aged mycelia of dry thread Pyrenomycetes for Trichoderma LTR-2, the propagation rate that shows as Trichoderma bacterium colony covering dry thread Pyrenomycetes bacterium colony slows down and then stops, and microscopically mycelia parasitic rate obviously descends.The results are shown in Figure 5.
(7) root colonization of green tricoderma LTR-2-2 test
The cotton that sowing shows money or valuables one carries unintentionally in healthy soil (cotton No. 12 of Shandong) seed is 10 with bacteria containing amount
8The green tricoderma LTR-2 of cfu/mL-2 conidial suspension soaks seed 30min, and it is thick to cover same soil 2cm, cultivates in 20-30 ℃ greenhouse.After 30 days, take out cotton plant carefully, measure fungi content in cotton rhizosphere and the surrounding soil respectively, calculate both ratios (R/S), as the index of fungi rhizosphere colonization ability with the PDA substratum that contains paraxin 100 μ g/mL.With the soil of being close to the root table as the rhizosphere part, with from plant main root 3-5cm, from soil table 2cm, and the soil that does not have a fibrous root as around (non-rhizosphere) soil.Be contrast with the cotton that does not inoculate Trichoderma during counting, the background correction fungi.The results are shown in Table 5.
The colonization ability (* 10 of table 5 green tricoderma LTR-2-2 in cotton rhizosphere and soil
3Cfu/g)
| Rhizosphere colony | Polypedon | The R/S value |
| 4310 | 1.0 | 4310 |
5, the application of green tricoderma LTR-2-2
(1) application of green tricoderma LTR-2 of the present invention-2 on agricultural is that effective constituent is mixed with Agrotechnical formulation with the spore of living, a kind of wettable powder or water dispersible microgranules, and component is as follows, is weight part:
Green tricoderma LTR-2-2 spore powder 0.05-0.2 part
Starch or Semen Maydis powder 92.1-96.05 part
Xanthan gum 1.0-2.0 part
SODLUM FULVATE 0.1-0.2 part
Sodium dodecylbenzene sulfonate 2.5-5.0 part
Chitin powder 0.3-0.5 part.
Said components is pulverized with supper micron mill, crossed 325 mesh sieves.
Said green tricoderma LTR-2-2 spore powder is the product of the solid fermentation cultural method step (5) of above-mentioned 3. green tricoderma LTR-2s-2.
Viride LTR-2 spore powder is effective sterilization component in the said components, and starch or Semen Maydis powder are dispersion agent, and xanthan gum is anti-dehydration, antiultraviolet, stablizer, emulsifying agent, wetting agent, Sodium dodecylbenzene sulfonate is a wetting agent, emulsifying agent, ultraviolet protecting agent, SODLUM FULVATE is a ultraviolet protecting agent, chitin provides nutrition for Trichoderma, induces Trichoderma to produce chitinase.
(2) application of green tricoderma LTR-2 of the present invention-2 on agricultural, extracting the antimicrobial component pyrone from spore is that effective constituent is mixed with Agrotechnical formulation, a kind of wettable powder, component is as follows, is weight part:
Diatomite 68-84 part
The enriched material 10-20 part that contains pyrone
Sodium dodecylbenzene sulfonate 5-10 part
Tween 80 1-2 part.
Said components is pulverized with supper micron mill, crossed 325 mesh sieves.
The said enriched material that contains pyrone is above-mentioned 4 (5) product.
Diatomite is carrier in the said components, and Sodium dodecylbenzene sulfonate is an auxiliary agent, and non-ionic surfactant Tween 80 is a defoamer.
Description of drawings
Fig. 1 is the dull and stereotyped form of cultivating of the PDA of green tricoderma LTR-2-2.
Fig. 2 is the conidiophore of green tricoderma LTR-2-2.
Fig. 3 is the restraining effect of green tricoderma LTR-2-2 pair cotton-wilt fusarium on agar plate, and the centre is the LTR-2 bacterium colony, and white portion is the cotton-wilt fusarium bacterium colony on every side.
Fig. 4 is that the microbiotic pyrone is to the dull and stereotyped restraining effect of the PDA of dry thread Pyrenomycetes.1 is contrast, and 2 is pyrone.
Fig. 5 is the supercrescence of green tricoderma LTR-2-2 pair dry thread Pyrenomycetes.3 is the mycelia of LTR-2, and 4 is the mycelia of dry thread Pyrenomycetes.The mycelia of LTR-2 is wrapped in above the mycelia of dry thread Pyrenomycetes.
Embodiment
Embodiment 1: the solid fermentation of green tricoderma LTR-2-2 is cultivated, and below is weight ratio or weight percent.
Green tricoderma LTR-2-2, preserving number is CGMCC NO.1498, and separation from soil, screening obtain, and find that through indoor flat plate antagonistic experiment and land for growing field crops biological control test this bacterial strain antimicrobial spectrum is wide, and prevention effect is good, and easily cultivation is pollution-free.
The solid fermentation of green tricoderma LTR-2-2 is cultivated, and carries out according to the following steps:
(1) slant strains: adopt solid PDA substratum, this green tricoderma LTR-2-2 is seeded on the test tube substratum, cultivated 2-3 days for 28 ℃.
(2) eggplant bottle bacterial classification: adopt liquid PDA substratum, test tube strains be seeded in the liquid eggplant bottle, place on the shaking table 28 ℃ shaking culture 3-4 days.
(3) liquid spawn: adopt seed culture medium, Semen Maydis powder 2%, glucose 0.5%, soybean cake powder 1%, dipotassium hydrogen phosphate 0.2%, potassium primary phosphate 0.3%, lime carbonate 1%, pH6.0 sterilized 40 minutes for 121 ℃, the seed of 1-2 eggplant bottle is washed with sterilized water, be inoculated in the seeding tank of 150 liters 30 ℃ of cultivations, air flow 1: 0.6-0.8, stirring velocity is 200r/min, and incubation time is 36-48 hour.
(4) solid spawn: adopt solid medium, wheat bran: rice husk: Semen Maydis powder=7: 1: 2, water content is 70% (comprising the moisture of the liquid spawn that inoculates), pH6.0,121 ℃ of sterilizations were cooled off with recirculated water after 40 minutes, in the combined inoculation device liquid spawn and solid medium are mixed, inoculum size is 5-10%, and inoculation finishes and transfers to the solid culture indoor cultivation.
(5) cultivate: substratum thickness is 5cm, and the material temperature control is at 30 ± 2 ℃, and room temperature is controlled at 25-30 ℃ ± 2 ℃, and the relative water content of air is controlled at 95-100%, and incubation time is 5-7 days.
Cultivation finishes, and with the solid culture natural air drying, the finished product water content is controlled at 5-10%, gets green tricoderma LTR-2-2 solid culture.Cross 100 mesh sieves and collect spore powder.
Embodiment 2: the application of green tricoderma LTR-2-2
Green tricoderma LTR-2-2 Agrotechnical formulation, it is as follows to fill a prescription, and is weight part:
0.05 part of LTR-2 spore powder
96.05 parts of starch or Semen Maydis powder
1.0 parts of xanthan gum
0.1 part of SODLUM FULVATE
2.5 parts of Sodium dodecylbenzene sulfonatees
0.3 part in chitin powder.
Above-mentioned LTR-2 spore powder is the product of embodiment 1.
Embodiment 3: the application of green tricoderma LTR-2-2
Green tricoderma LTR-2-2 Agrotechnical formulation, it is as follows to fill a prescription, and is weight part:
0.2 part of LTR-2 spore powder
92.1 parts of starch or Semen Maydis powder
2.0 parts of xanthan gum
0.2 part of SODLUM FULVATE
5.0 parts of Sodium dodecylbenzene sulfonatees
0.5 part in chitin powder.
Above-mentioned LTR-2 spore powder is the product of embodiment 1.
Embodiment 4: the application of green tricoderma LTR-2-2
The Agrotechnical formulation of microbiotic pyrone is weight part:
84 parts in diatomite
10 parts of enriched materials that contain pyrone
5 parts of Sodium dodecylbenzene sulfonatees
1 part of tween 80.
The above-mentioned enriched material preparation method who contains pyrone is as follows, cultivates green tricoderma LTR-2-2 with the solid bran mass, obtains spore powder.Soaked spore powder 2 days with methylene dichloride 5-10 ℃, soak solution is successively through 5% charcoal absorption, the washing of 3% sodium carbonate solution, anhydrous sodium sulfate dehydration, at last treatment solution is evaporated to thickly, obtains containing the enriched material of pyrone, adopt the dull and stereotyped diffusion process of PDA to measure its bacteriostatic activity.The antibacterial band of result is 6.5mm.
Wherein, the preservation of biomaterial green tricoderma LTR-2-2 sample, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City; Preservation date: on October 20th, 2005; Deposit number: CGMCC No.1498; Classification name: viride (Trichoderma viride).
Claims (4)
1. viride bacterial strain LTR-2, preserving number is CGMCC NO.1498, feature description is as follows:
Bacterium colony is grown on the PDA flat board soon, and subiculum is thicker, fine and close clump pencil, initial stage is white, and is smooth, and the later stage is deep green because of producing conidium, produce the spore district and often be arranged in the concentric wheel stripe shape, the bacterium colony back side is colourless, is light yellow sometimes, mycelia is transparent, have every, cell walls is smooth, conidiophore is uprightly born by mycelia, and is colourless, and branch is many, to giving birth to or two to three grades of branches of alternate, integral body resembles branch; Branch and conidiophore approximate right angle, end is a stigma, the stigma doleiform, conidium sphere or oblong, surface irregularity perhaps is covered with little furcella; Unit cell leans on mucus to be gathered into the conidial head of spherical green on stigma.
2. the solid fermentation cultural method of claim 1 described green tricoderma LTR-2-2 carries out according to the following steps, is weight ratio:
(1) slant strains: adopt solid PDA substratum, this green tricoderma LTR-2-2 is seeded on the test tube substratum, cultivated 2-3 days for 28 ℃;
(2) eggplant bottle bacterial classification: adopt liquid PDA substratum, test tube strains be seeded in the liquid eggplant bottle, place on the shaking table 28 ℃ shaking culture 3-4 days;
(3) liquid spawn: adopt seed culture medium, Semen Maydis powder 2%, glucose 0.5%, soybean cake powder 1%, dipotassium hydrogen phosphate 0.2%, potassium primary phosphate 0.3%, lime carbonate 1%, pH6.0 sterilized 40 minutes for 121 ℃, the seed of 1-2 eggplant bottle is washed with sterilized water, be inoculated in the seeding tank of 150 liters 30 ℃ of cultivations, air flow 1: 0.6-0.8, stirring velocity is 200r/min, and incubation time is 36-48 hour;
(4) solid spawn: adopt solid medium, wheat bran: rice husk: Semen Maydis powder=7: 1: 2, water content is 70%, moisture comprising the liquid spawn that inoculates, pH6.0,121 ℃ of sterilizations were cooled off with recirculated water after 40 minutes, in the combined inoculation device liquid spawn and solid medium were mixed, inoculum size is 5-10%, and inoculation finishes and transfers to the solid culture indoor cultivation.
(5) cultivate: substratum thickness is 5cm, and the material temperature control is at 30 ± 2 ℃, and room temperature is controlled at 25-30 ℃ ± 2 ℃, and the relative water content of air is controlled at 95-100%, and incubation time is 5-7 days;
Cultivation finishes, and with the solid culture natural air drying, the finished product water content is controlled at 5-10%, gets green tricoderma LTR-2-2 solid culture, crosses 100 mesh sieves and collects spore powder.
3. the application of claim 1 described green tricoderma LTR-2-2, the spore powder of collecting from its solid culture, and be that effective constituent is mixed with Agrotechnical formulation with the spore of living, a kind of wettable powder or water dispersible microgranules, component is as follows, is weight part:
Green tricoderma LTR-2-2 spore powder 0.05-0.2 part
Starch or Semen Maydis powder 92.1-96.05 part
Xanthan gum 1.0-2.0 part
SODLUM FULVATE 0.1-0.2 part
Sodium dodecylbenzene sulfonate 2.5-5.0 part
Chitin powder 0.3-0.5 part,
Said components is pulverized with supper micron mill, crossed 325 mesh sieves.
4. the application of the described viride bacterial strain of claim 1 LTR-2, extracting the microbiotic pyrone from spore is that effective constituent is mixed with Agrotechnical formulation:
The extracting method of described microbiotic pyrone is: cultivate green tricoderma LTR-2-2 with solid medium, obtain spore powder, soaked spore powder 2 days with methylene dichloride 5-10 ℃, soak solution is successively through 5% charcoal absorption, the washing of 3% sodium carbonate solution, anhydrous sodium sulfate dehydration, at last treatment solution is evaporated to thickly, obtains containing the enriched material of microbiotic pyrone, prepare a kind of wettable powder with this enriched material, component is as follows, is weight part:
Diatomite 100-200 part
The enriched material 10-20 part that contains pyrone
Sodium dodecylbenzene sulfonate 5-10 part
Tween 80 1-2 part
Said components is pulverized with supper micron mill, crossed 325 mesh sieves.
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| US4489161A (en) * | 1982-10-25 | 1984-12-18 | The United States Of America As Represented By The Secretary Of Agriculture | Strain of Trichoderma viride to control fusarium wilt |
| US5192686A (en) * | 1987-07-10 | 1993-03-09 | Ahmad Syed J | Rhizosphere-competent trichoderma strains |
| CN1451635A (en) * | 2002-04-18 | 2003-10-29 | 孙连壮 | Production of fungus compound fertilizer for controlling plant insect pest and diseases by liquid fermentation and producing process thereof |
| CN1422946A (en) * | 2002-09-24 | 2003-06-11 | 宁夏农林科学院植物保护研究所 | Trichoderma strain for preventing and treating plant soil root infected disease, and fermentation and grain preparation process thereof |
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