CN104059860A - Trichoderma koningiopsis TZ-11 bacterial strain and application thereof - Google Patents
Trichoderma koningiopsis TZ-11 bacterial strain and application thereof Download PDFInfo
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- CN104059860A CN104059860A CN201410316956.2A CN201410316956A CN104059860A CN 104059860 A CN104059860 A CN 104059860A CN 201410316956 A CN201410316956 A CN 201410316956A CN 104059860 A CN104059860 A CN 104059860A
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Abstract
The invention relates to a Trichoderma koningiopsis TZ-11 bacterial strain, an application and a method for preparing a preparation of the Trichoderma koningiopsis TZ-11 bacterial strain. According to the bacterial strain, the wettable powder prepared by conducting solid fermentation can be used for preventing and treating the anthracnose, caused by infections of Glomerella cingulata, of many crops such as pepper, mango, banana and loquat and especially for preventing and treating the grape anthracnose, the strawberry anthracnose and other plant diseases caused by infection of Glomerella cingulata. The field test result shows that the fungicide has a good preventing and controlling effect on the grape anthracnose and the strawberry anthracnose, the bacterial strain fermenting technology is simple, and the production cost is low. The fungicide living spore content is high, and the storage quality is stable.
Description
Technical field
The present invention relates to strain microorganism strains and uses thereof, the preparation method more specifically to a strain trichoderma pseudokiningii (Trichoderma koningiopsis) TZ-11 bacterial strain, purposes and preparation thereof, belongs to biological pesticide technical field.
Background technology
The anthrax of being enclosed grape, apple, pears and the strawberry etc. of small cluster shell Pseudomonas (Glomerellaspp.) initiation by plant pathogenic fungi is the Major Diseases of industrialized agriculture farm crop.Chemical agent prevention and control are current main prophylactico-therapeutic measuress that prevent and treat anthrax.Conventional chemical agent mainly contains derosal (carbendazim), Wocosin 50TK (propiconazole), Bitertanol (bitertanol), imazalil (imazalil), own azoles alcohol (hexaconazole), Azoxystrobin (azoxystrobin), kresoxim-methyl (kresoxim-methyl), phonetic mould amine (pyrimethanil), difenoconazole (difenoconazole), enostroburin (enestroburin), prochloraz (prochloraz) and pyraclostrobin (pyraclostrobin) etc.Peasant household is mostly used above-mentioned high-efficiency low-toxicity medicament in anthracnose of crop control, but such pharmacy effect site single (suppressing the transmission of mitochondrial respiratory chain electronics).Under height is prevented and treated the frequency, very easily develop immunity to drugs, and between medicament, mutual resistance risk is high.In production, occur repeatedly the example that control is failed, illustrate that Anthracnose Pathogen fungi has all produced anti-(resistance to) property of medicine in various degree to above-mentioned main chemical agent, the research and development that substitute medicament are very urgent.
With respect to field crop biological control of diseases, the biological control of the fruits in season diseases such as grape, strawberry research just just starts in China.Also do not have successfully for preventing and treating the biological pesticide special preparation of anthrax both at home and abroad at present.The research of having reported mostly concentrates on and utilizes living microorganism to carry out prevention and control, as utilize the viride (Trichoderma viride) in trichoderma fungi (Trichoderma spp.), trichoderma harziarum (Trichoderma harzianum) and long shoot wood mould (Trichoderma longibrachiatum), subtilis (Bacillus subtilis) in bacillus (Bacillus spp.), waxy Bacillus (Bacillus cereus), bacillus polymyxa (Bacillus polymyxa), bacillus megaterium (Bacillus megaterium) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens), pseudomonas cepacia (Pseudomonas cepacia) in Rhodopseudomonas bacterium (Pseudomonas spp.), the false single-cell bacteria (Pseudomonas fluorescens) of fluorescence and pseudomonas putida (Pseudomonas putida), also cover pichia (Pichia guilliermondii) and Cryptococcus laurentii (Cryptococcus laurentii) season in yeast, pythium oligandrum (Pythium oligandrum) etc. is prevented and treated.The research report that uses living microorganism to prevent and treat anthracnose of crop only limits to toxicity mensuration, greenhouse control test and field test mensuration stage.The trichoderma pseudokiningii bacterial strain that can effectively prevent and treat anthrax provided by the invention, does not mention in the prior art and discloses.
Summary of the invention
The first object of the present invention is to provide a kind of and can be used for control by the bacterial strain that encloses small cluster shell bacterium (Glomerella cingulata) and infect the anthrax disease causing.
The second object of the present invention is to provide bacterial strain and by enclosing small cluster shell bacterium (Glomerella cingulata), infects the application causing aspect anthrax disease in control, especially infects the bitter rot or anthracnose of grape that causes and the application on Strawberry anthracnose enclosing small cluster shell bacterium.
The 3rd object of the present invention is a kind of wettable powder that utilizes above-mentioned bacterial strains to prepare.
The 4th object of the present invention is to provide a kind of preparation method of above-mentioned wettable powder.
One, for solving the first object, the technical scheme providing is as follows:
One strain trichoderma pseudokiningii bacterial strain, it is characterized in that, the Classification And Nomenclature of this bacterial strain is trichoderma pseudokiningii TZ-11 (Trichoderma koningiopsisTZ-11), for this bacterial strain on July 8th, 2010 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is: CGMCCNo.3969.
Morphology and the microscopic features of bacterial strain of the present invention are as follows:
Fast in the upper growth of wort agar substratum (MEA), under 25 ℃ of dark conditions, cultivate 4d colony diameter 60-63mm, initial stage white, the later stage is greyish-green because producing spore, and quality is cotton-shaped; The back side is light brown, without water colo(u)r.Conidium structure forms in a large number, and Chan Bao district is uniformly distributed.Mycelia wall is level and smooth or coarse, multi-branched, and tool is separated, wide 2.0-3.5 μ m; Conidiophore wall is smooth, and main shaft is obvious, less to branch situation estranged, wide 2.0-3.5 μ m; Product spore bottle obstructs most 2-4 and clusters on conidiophore or its branch, base portion column or 6.2-15.0 * 2.0-3.5 μ m that slightly expands; Conidium is oval, deep green, and wall is level and smooth or slightly coarse, 3.5-5.5 * 1.8-3.0 μ m.
DNA sequence dna (this section of rRNA comprised 18SrRNA fragment, ITS1,5.8SrRNA, ITS2 district complete sequence and 28SrRNA sequence fragment) corresponding to trichoderma pseudokiningii of the present invention (Trichoderma koningiopsis) TZ-11 bacterial strain rRNA gene order is as shown in SEQ ID NO:1.
Two, bacterial strain infects the application aspect the anthrax disease causing in control by enclosing small cluster shell bacterium (Glomerellacingulata), especially the application in enclosing the microbial bitter rot or anthracnose of grape of small cluster shell and Strawberry anthracnose control.
Three, a kind of wettable powder that utilizes above-mentioned bacterial strains to prepare.
Four, a preparation method for above-mentioned wettable powder, specifically comprises the following steps:
(A) acquisition of solid fermentation culture
(1) after wheat bran and wood sawdust are mixed in the ratio of 3:7 (V/V), the sucrose and the volatile salt that add respectively 0.1% (W/W), and adding water, to make water content be 60～65%, pH is adjusted to 7.5-8.0, after again mixing thickening polyethylene plastic bag in every bag of packing 1.5kg, tighten sack, and at 121 ℃ moist heat sterilization 60min.At 2 sterilizing newspapers of level trough solid fermentation tray bottom place mat, add the solid fermentation substratum 1.5kg after sterilizing, charging thickness is about 5cm.By the trichoderma pseudokiningii TZ-11 bacterial strain being transferred in advance in PDA plate, be placed in interior 28 ℃ of incubator and cultivate after 7d, with the spore under aseptic washing, with after microscopic counting, spore liquid concentration being allocated as: 1.0 * 10
7individual/mL.This spore liquid of 100mL is evenly inoculated into after 1.5kg solid fermentation substratum, at 2 sterilizing newspapers of fermentation dish top cover and tighten sealing with jag, finally be positioned over fermentation in 28 ℃ of moisturizing incubators, yeasting relative humidity reaches more than 80%, fermentation 8-10d, tunning spore amount alive can reach 1.0-2.0 * 10
9cfu/g;
(2) above-mentioned tunning is added appropriate distilled water be placed in 150rpm vibration washing 8min on shaking table, then with 325 mesh standard sieves, filter and collect filtrate, 3 times repeatedly, finally, by filtrate centrifugal 15min under 3000rpm, collecting precipitation thing is standby.
(B) microbial inoculum complete processing
(1) will collect through solid fermentation, after centrifugal 0.5% (W/W) methocel solution (solution is prepared with sterilized water) for throw out and suspend, and the content of the spore of living in suspension is adjusted to>=5.0 * 10
8cfu/mL;
(2) sodium metnylene bis-naphthalene sulfonate, calcium dodecylbenzene sulphonate, light calcium carbonate and diatomite are pressed after the weight ratio mixing of 5:4:1:90, by comminution by gas stream, make more than fineness reaches 325 orders, then add the spore suspension preparing, finally by 50 ℃ of airflow dryings, stirrers, mix and pack post-treatment and become containing the live wettable powder of spore of trichoderma pseudokiningii TZ-11 bacterial strain.Spore content>=1.0 * 10 alive of wherein said trichoderma pseudokiningii TZ-11 bacterial strain wettable powder
8cfu/g.
Beneficial effect of the present invention
(1) the present invention is in order to solve the problem of anthrax control in the arable farmings such as grape and strawberry, in conjunction with agriculture production actual demand, provide a strain new biocontrol strain---trichoderma pseudokiningii TZ-11 bacterial strain, the wettable powder being made through solid fermentation by this bacterial strain, can be used for control and infect by enclosing small cluster shell bacterium (Glomerella cingulata) anthrax causing, capsicum for example, mango, the control of a lot of anthracnose of crop such as banana and loquat, especially by enclosing small cluster shell bacterium (Glomerella cingulata), infect the diseases such as the bitter rot or anthracnose of grape that causes and Strawberry anthracnose.Field test results shows, this microbial inoculum all has good prevention effect to bitter rot or anthracnose of grape and Strawberry anthracnose.
(2) strain fermentation process is simple, low production cost; Microbial inoculum spore content alive is high, and storing property is stable.
Trichoderma pseudokiningii TZ-11 of the present invention (Trichoderma koningiopsis TZ-11) bacterial strain is in China Committee for Culture Collection of Microorganisms common micro-organisms center (abbreviation CGMCC July 8 in 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica) preservation, deposit number is: CGMCCNo.3969.
Embodiment
The present invention is further described to enumerate embodiment below, but therefore do not limit content of the present invention.
Embodiment 1 the present embodiment explanation obtains the method for trichoderma pseudokiningii (Trichoderma koningiopsis) TZ-11 bacterial strain.
PDA culture medium prescription is: potato (peeling) 200g/L, glucose 20g/L, water 1L, agar 20g/L, pH nature.By peeling potatoes, be cut into small pieces, in pot, add the about 1L of water and boil half hour, with double gauze, filter, get its filtrate and add glucose, and add water and supply 1L.
Comprise the following steps:
1) separation of bacterial strain, purifying and preservation.
Collected specimens on kyoto grape in grape growing region, Jurong City, Jiangsu Province, the sample of collection is carried out on PDA plate to separate tissue on Bechtop, according to the timely picking different shape of the colony characteristics of fungi list bacterium colony to PDA plate, purifying 2 times, record bacterial strain number, the bacterial strain of purifying is forwarded to and on PDA inclined-plane, cultivates that to put into 4 ℃ of refrigerators after good standby.Then will in above-mentioned separated fungal bacterial strain switching of preserving, on PDA plate, activate, adopt the screening of plate face-off culture method to enclose the antagonistic strain of small cluster shell bacterium (Glomerella cingulata), according to the antagonistic activity of the size evaluation bacterial strain of inhibition zone.
2) identify bacterial strain kind preservation.
After the strongest strain passage purifying of antagonistic activity is cultivated, bacterial strain is carried out to Molecular Identification, qualification result shows that bacterial strain is trichoderma pseudokiningii (Trichoderma koningiopsis), to its called after trichoderma pseudokiningii (Trichoderma koningiopsis) TZ-11 bacterial strain, and make trichoderma pseudokiningii (Trichoderma koningiopsis) the lyophilize pipe of TZ-11 bacterial strain and carry out the cold storage of original strain.
The colonial morphology of above-mentioned bacterial classification and microscopic features are: fast in the upper growth of wort agar substratum (MEA), under 25 ℃ of dark conditions, cultivate 4d colony diameter 60-63mm, and initial stage white, the later stage is greyish-green because producing spore, and quality is cotton-shaped; The back side is light brown, without water colo(u)r.Conidium structure forms in a large number, and Chan Bao district is uniformly distributed.Mycelia wall is level and smooth or coarse, multi-branched, and tool is separated, wide 2.0-3.5 μ m; Conidiophore wall is smooth, and main shaft is obvious, less to branch situation estranged, wide 2.0-3.5 μ m; Product spore bottle obstructs most 2-4 and clusters on conidiophore or its branch, base portion column or 6.2-15.0 * 2.0-3.5 μ m that slightly expands; Conidium is oval, deep green, and wall is level and smooth or slightly coarse, 3.5-5.5 * 1.8-3.0 μ m.
DNA sequence dna (this section of rRNA comprised 18SrRNA fragment, ITS1,5.8SrRNA, ITS2 district complete sequence and 28SrRNA sequence fragment) corresponding to trichoderma pseudokiningii of the present invention (Trichoderma koningiopsis) TZ-11 bacterial strain rRNA gene order is as shown in SEQIDNO:1.。
The embodiment 2 use bacterial strain TZ-11 that prevents and treats anthrax of the present invention prepares wettable powder
Preparation method comprises the following steps:
(A) acquisition of solid fermentation culture
(1) after wheat bran and wood sawdust are mixed in the ratio of 3:7 (V/V), the sucrose and the volatile salt that add respectively 0.1% (W/W), and adding water, to make water content be 60～65%, pH is adjusted to 7.5-8.0, after again mixing thickening polyethylene plastic bag in every bag of packing 1.5kg, tighten sack, and at 121 ℃ moist heat sterilization 60min.At 2 sterilizing newspapers of level trough solid fermentation tray bottom place mat, add the solid fermentation substratum 1.5kg after sterilizing, charging thickness is about 5cm.By the trichoderma pseudokiningii TZ-11 bacterial strain being transferred in advance in PDA plate, be placed in interior 28 ℃ of incubator and cultivate after 7d, with the spore under aseptic washing, with after microscopic counting, spore liquid concentration being allocated as: 1.0 * 10
7individual/mL.This spore liquid of 100mL is evenly inoculated into after 1.5kg solid fermentation substratum, at 2 sterilizing newspapers of fermentation dish top cover and tighten sealing with jag, finally be positioned over fermentation in 28 ℃ of moisturizing incubators, yeasting relative humidity reaches more than 80%, fermentation 8-10d, tunning spore amount alive can reach 1.0-2.0 * 10
9cfu/g; (2) above-mentioned tunning is added appropriate distilled water be placed in 150rpm vibration washing 8min on shaking table, then with 325 mesh standard sieves, filter and collect filtrate, 3 times repeatedly, finally, by filtrate centrifugal 15min under 3000rpm, collecting precipitation thing is standby.
(B) microbial inoculum complete processing
(1) will collect through solid fermentation, after centrifugal 0.5% (W/W) methocel solution (solution is prepared with sterilized water) for throw out and suspend, and the content of the spore of living in suspension is adjusted to>=5.0 * 10
8cfu/mL; (2) sodium metnylene bis-naphthalene sulfonate, calcium dodecylbenzene sulphonate, light calcium carbonate and diatomite are pressed after the weight ratio mixing of 5:4:1:90, by comminution by gas stream, make more than fineness reaches 325 orders, then add the spore suspension preparing, finally by 50 ℃ of airflow dryings, stirrers, mix and pack post-treatment and become containing the live wettable powder of spore of trichoderma pseudokiningii TZ-11 bacterial strain.Spore content>=1.0 * 10 alive of wherein said trichoderma pseudokiningii TZ-11 bacterial strain wettable powder
8cfu/g.
Implement 3 test example
(1) TZ-11 bacterial strain wettable powder is to enclosing the indoor virulence of small cluster shell bacterium (Glomerella cingulata)
TZ-11 bacterial strain wettable powder is as follows to enclosing the Toxicity Determination method of small cluster shell bacterium:
Respectively with sterilized water by 1.0 * 10
8cfu/gTZ-11 bacterial strain wettable powder and 1.0 * 10
6cfu/g pythium oligandrum wettable powder (Bi Aorui bio tech ltd, Beijing) is diluted to finite concentration successively, again 1mL liquid and 9mLPDA substratum are mixed in culture dish, make the PDA substratum containing serial gradient concentration medicament, the serial gradient concentration of each medicament in PDA substratum is all designed to 1.0 * 10
5, 5.0 * 10
4, 1.0 * 10
4, 5.0 * 10
3, 1.0 * 10
3, 5.0 * 10
2with 1.0 * 10
2cfu/mL, adopts sterilized water to compare, and each is processed and repeats 4 times.The small cluster shell bacterium that encloses retaining is transplanted in PDA plate, at 25 ℃, activate 96h, then at nearly colony edge, with punch tool, produce the bacterium cake that diameter is 5mm, and be transplanted in the PDA plate of above-mentioned pastille and blank, cultivate 96h for 25 ℃, in contrasting, bacterium colony grows to 4/5 o'clock of about plate diameter, adopts right-angled intersection method to measure colony diameter.Calculate colony diameter mean value, and calculate the average inhibiting rate of mycelial growth according to following formula: the average inhibiting rate of mycelial growth={ (contrast colony diameter average-processing colony diameter average)/(contrast colony diameter average-inoculation bacterium cake diameter) } * 100%.Adopt DPS data handling system, calculate regression equation, EC that each medicament suppresses enclosing small cluster shell bacterium mycelial growth
50and 95% fiducial limit.
TZ-11 bacterial strain wettable powder is to enclosing the Toxicity Determination result of small cluster shell bacterium:
Toxicity Determination result (table 1) shows, the EC that TZ-11 bacterial strain wettable powder and pythium oligandrum wettable powder suppress enclosing small cluster shell bacterium mycelial growth
50be respectively: 8.2979 * 10
2with 1.4434 * 10
3μ g/mL, differs approximately 1.7 times, TZ-11 bacterial strain wettable powder to the activity of enclosing small cluster shell bacterium mycelial growth and suppressing higher than pythium oligandrum wettable powder.
The wettable powder of table 1TZ-11 bacterial strain is to enclosing the Toxicity Determination result of small cluster shell bacterium (Glomerella cingulata)
(2) field control effectiveness test of TZ-11 bacterial strain wettable powder to bitter rot or anthracnose of grape
Test treatment process: test site is positioned at vineyard, Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute, and kind is huge peak, the cultivation of horizontal rack formula, the age of tree approximately 20 years.Soil is horse liver soil, middle fertility, and organic content 1.75%, duration of test orchard management routine.Test is established: TZ-11 bacterial strain wettable powder 1000 and 2000 times of liquid, 8000 times of liquid of pythium oligandrum wettable powder (be recommended dose, need 30 minutes in advance preparation stoste before spraying), and take clear water spraying as contrast, spray amount is 900kg/hm
2, each community area 100m
2, random district group is arranged, and repeats for 4 times.Test in the 1st spray medicine on June 3, every 10d control once, prevent and treat altogether 5 times later.After 5 medicines, 14d investigates the incidence of each community, during investigation, in each community, select at random 4 strain grapes, in upper, middle and lower, orientation, 5 of left and rights gets respectively 1 fruit ear, totally 20 fruit ears, record in fruit ear axle, fruit grain and carpopodium disease a situation arises, calculate disease and refer to and prevention effect.
Field controling test result (table 2) shows, TZ-11 bacterial strain wettable powder 1000 and 2000 times of liquid are respectively 88.08% and 73.37% to the preventive effect of bitter rot or anthracnose of grape, significant difference; 8000 times of liquid of pythium oligandrum wettable powder are 80.27% to the preventive effect of bitter rot or anthracnose of grape, and preventive effect is lower than 1000 times of liquid of TZ-11 bacterial strain wettable powder, significant difference.
The field control effectiveness test result of table 2TZ-11 bacterial strain wettable powder to bitter rot or anthracnose of grape
Note: the different conspicuous level (p<0.05) that reaches of different lowercase alphabet differentials after data
(3) field control effectiveness test of TZ-11 bacterial strain wettable powder to Strawberry anthracnose
Test treatment process: test site is positioned at the strawberry Demonstration Garden of white rabbit town, Jurong City, Jiangsu Province cloud rabbitweed certain kind of berries cooperative society, and kind is red cheek.Test is established: TZ-11 bacterial strain wettable powder 1000 and 2000 times of liquid, 8000 times of liquid of pythium oligandrum wettable powder (be recommended dose, need 30 minutes in advance preparation stoste before spraying), and take clear water spraying as contrast, spray amount is 900kg/hm
2, each community area 50m
2, random district group is arranged, and repeats for 4 times.Test in the 1st spray medicine on May 3rd, 2013, every 10d control once, prevent and treat altogether 6 times later.After 6 medicines, 14d investigates the incidence of each community, and during investigation, in sampling survey approximately 100 strains of 5 of each communities, a situation arises to record every strain Strawberry anthracnose, calculates disease and refers to and prevention effect.
Field controling test result (table 3) shows, TZ-11 bacterial strain wettable powder 1000 and 2000 times of liquid are respectively 85.63% and 69.92% to the preventive effect of Strawberry anthracnose, significant difference; 8000 times of liquid of pythium oligandrum wettable powder are 80.38% to the preventive effect of Strawberry anthracnose, and preventive effect is lower than 1000 times of liquid of TZ-11 bacterial strain wettable powder, significant difference.
The field control effectiveness test result of table 3TZ-11 bacterial strain wettable powder to Strawberry anthracnose
Note: the different conspicuous level (p<0.05) that reaches of different lowercase alphabet differentials after data.
Sequence table
<110> Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute
<120> mono-strain trichoderma pseudokiningii TZ-11 bacterial strain and uses thereof
<130> 1
<160> xb14070401
<170> PatentIn version 3.3
<210> 1
<211> 564
<212> DNA
<213> trichoderma pseudokiningii
<400> 1
gaggtcaaca tttcagaaag ttgggtgttt tacggacgtg gacgcgccgc gctcccggtg 60
cgagttgtgc aaactactgc gcaggagagg ctgcggcgag accgccactg tatttcgggg 120
ccgggatccc gtcttagggg ttcccgatcc ccaacgccga ccccccggag gggttcgagg 180
gttgaaatga cgctcggaca ggcatgcccg ccagaatact ggcgggcgca atgtgcgttc 240
aaagattcga tgattcactg aattctgcaa ttcacattac ttatcgcatt tcgctgcgtt 300
cttcatcgat gccagaacca agagatccgt tgttgaaagt tttgattcat tttgaatttt 360
tgctcagagc tgtaagaaat aacgtccgcg aggggactac agaaagagtt tggttggtcc 420
ctccggcggg cgcctggttc cggggctgcg acgcacccgg ggcgtgaccc cgccgaggca 480
acagtttggt atggttcaca ttgggtttgg gagttgtaaa ctcggtaatg atccctccgc 540
tggttcacca acggagacct tgtt 564
Claims (6)
1. a strain trichoderma pseudokiningii bacterial strain, is characterized in that, the Classification And Nomenclature of this bacterial strain be trichoderma pseudokiningii TZ-11 (
trichoderma koningiopsistZ-11), this bacterial strain on July 8th, 2010 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is: CGMCC No.3969.
2. bacterial strain according to claim 1, is characterized in that, shown in SEQ ID NO:1 is DNA sequence dna corresponding to rRNA gene order, and this section of rRNA comprised 18S rRNA fragment, ITS1,5.8S rRNA, ITS2 district complete sequence and 28S rRNA sequence fragment.
Bacterial strain described in claim 1-2 control by enclose small cluster shell bacterium (
glomerella cingulata) application of infecting the anthrax disease aspect causing.
4. a wettable powder of preparing with the bacterial strain described in claim 1-2.
5. a method of preparing wettable powder according to claim 4, is characterized in that comprising the following steps:
(A) acquisition of solid fermentation culture
(1) after wheat bran and wood sawdust are mixed in the ratio of 3:7 (V/V), the sucrose and the volatile salt that add respectively 0.1% (W/W), and adding water, to make water content be 60 ~ 65%, pH is adjusted to 7.5-8.0, after again mixing thickening polyethylene plastic bag in every bag of packing 1.5kg, tighten sack, and at 121 ℃ moist heat sterilization 60 min;
At 2 sterilizing newspapers of level trough solid fermentation tray bottom place mat, add the solid fermentation after sterilizing to cultivate based 1.5 kg, charging thickness is about 5 cm;
By the trichoderma pseudokiningii TZ-11 bacterial strain being transferred in advance in PDA plate, be placed in interior 28 ℃ of incubator and cultivate after 7d, with the spore under aseptic washing, with after microscopic counting, spore liquid concentration being allocated as: 1.0 * 10
7individual/mL;
This spore liquid of 100mL is evenly inoculated into after 1.5 kg solid fermentation substratum, at 2 sterilizing newspapers of fermentation dish top cover and tighten sealing with jag, finally be positioned over fermentation in 28 ℃ of moisturizing incubators, yeasting relative humidity reaches more than 80%, fermentation 8-10d, tunning spore amount alive can reach 1.0-2.0 * 10
9cfu/g;
(2) above-mentioned tunning is added appropriate distilled water be placed in 150rpm vibration washing 8min on shaking table, then with 325 mesh standard sieves, filter and collect filtrate, 3 times repeatedly, finally, by filtrate centrifugal 15min under 3000rpm, collecting precipitation thing is standby;
(B) microbial inoculum complete processing
(1) will collect through solid fermentation, after centrifugal 0.5% (W/W) methocel solution for throw out and suspend, wherein solution is prepared with sterilized water, and the content of the spore of living in suspension is adjusted to>=5.0 * 10
8cfu/mL;
(2) sodium metnylene bis-naphthalene sulfonate, calcium dodecylbenzene sulphonate, light calcium carbonate and diatomite are pressed after the weight ratio mixing of 5:4:1:90, by comminution by gas stream, make more than fineness reaches 325 orders, then add the spore suspension preparing, finally by 50 ℃ of airflow dryings, stirrers, mix and pack post-treatment and become containing the live wettable powder of spore of trichoderma pseudokiningii TZ-11 bacterial strain.
6. according to the preparation method of the wettable powder described in claim 5, it is characterized in that described trichoderma pseudokiningii TZ-11 bacterial strain spore content>=1.0 * 10 alive
8cfu/g.
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| CN107372617A (en) * | 2017-08-07 | 2017-11-24 | 聊城大学 | Trichoderma pseudokiningii bacterium wettable powder and preparation method thereof |
| CN108373978A (en) * | 2018-02-12 | 2018-08-07 | 淄博职业学院 | A kind of microorganism formulation and its application |
| CN105255949B (en) * | 2015-11-09 | 2018-08-17 | 东北林业大学 | A kind of small-scale Trichoderma process for solid state fermentation |
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| CN112143654A (en) * | 2020-09-08 | 2020-12-29 | 华南农业大学 | Trichoderma pseudokoningii and application thereof in preventing and treating litchi anthracnose |
| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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| CN112143654B (en) * | 2020-09-08 | 2022-03-22 | 华南农业大学 | Trichoderma pseudokoningii and application thereof in preventing and treating litchi anthracnose |
| CN114933974A (en) * | 2022-05-11 | 2022-08-23 | 华北理工大学 | Trichoderma pseudokoningii C5-9 and application thereof in antagonism of botrytis cinerea |
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