CN106434370A - Trichoderma protoplast fusion strain and application thereof - Google Patents
Trichoderma protoplast fusion strain and application thereof Download PDFInfo
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- CN106434370A CN106434370A CN201610847352.XA CN201610847352A CN106434370A CN 106434370 A CN106434370 A CN 106434370A CN 201610847352 A CN201610847352 A CN 201610847352A CN 106434370 A CN106434370 A CN 106434370A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention relates to a trichoderma protoplast fusion strain and application thereof. The trichoderma protoplast fusion strain is a trichoderma fusant Tpf-2, wherein the preservation number is CGMCC No.12477, the preservation date is May 30, 2016, the preservation unit is China General Microbiological Culture Collection Center, and the address is 3#, yard No.1, Bechen west road, Chaoyang District, Beijing. The trichoderma protoplast fusion strain can effectively prevent and treat diseases caused by pythium aphanidermatum and pythium wltmum, the prevention and treatment effect on tomato damping-off caused by the pythium aphanidermatum and the pythium wltmum reaches 86.3 percent and 85.7 percent separately, the yield-increasing effect reaches 77.3 percent and 79.9 percent separately (p is less than 0.01), the survival rate of the trichoderma protoplast fusion strains which are colonized in tomato rhizosphere soil for five months reaches 75 percent, and the effective survival rate of chlamydospore preparations within 1-year guarantee period reaches 90 percent.
Description
Technical field
The present invention relates to a kind of Trichoderma spp. Protoplast fusant and its application are and in particular to one kind is gone out by heat-ultraviolet
Application in the rotten mould damping off preventing and treating of Fructus Lycopersici esculenti for the Trichoderma spp. fusant and this fusant that protoplast fusion of living obtains, belongs to raw
Thing technical field.
Background technology
Fructus Lycopersici esculenti is the main vegetable crop of China, and multiple diseases and insect pest often occur in planting process, and wherein Fructus Lycopersici esculenti is sudden
Disease of falling is the important disease of Fructus Lycopersici esculenti, multiple be born in seedling stage, lead to seedling folding to fall, its cause of disease predominantly pythium spp Pythium spp.
In the rotten mould P.aphanidermatum and Pythium ultimum P.ultimum of melon and fruit, such pathogenic bacteria is in oomycota Mastigomycotina
Pythium lower fungus, growth rate is fast, often in soil, in water saprophytic or parasitic survival for a long time, to agricultural production band
Carry out heavy losses.
Prevent and treat the microbial inoculum of Fructus Lycopersici esculenti damping off both at home and abroad mainly based on chemical agent, at present due to excessive and unreasonable use
A series of health and environmental security that chemical pesticide control disease is brought, the Ministry of Agriculture formulates《To the year two thousand twenty Pesticide use
Amount zero growth rate action scheme》, the green prevention and control technology such as application cultural control, Biological control, physical control are proposed, thus reaching few
The purpose of medication.Abroad only register two related microorganisms Pesticidal products with pythium oligandrum as effective ingredient, state at home
Related product is not inside had to register.
Trichoderma spp. Trichoderma spp. is worldwide distribution funguses, is primarily present in soil, rotten timber and weeds
In, because it obtains extensive concern and research in the using value of the aspects such as industrial enzyme preparation, antibiotic and Biological control.But by
Not ideal enough in the field efficacy of the Trichoderma preparation of natural source, constrain the application of its further genralrlization.
Protoplast fusion is the gene recombination technology that 20 century 70s grow up, and has many conventional hybridization methods
Incomparable original advantage, by preparing the protoplast of Trichoderma spp., and then carries out mutation or fusion, is that trichoderma strain is carried out
The effective means of improvement selection-breeding.The more traditional biofungicide of product after this improvement will have higher biological and ecological methods to prevent plant disease, pests, and erosion efficiency and more
The strong market competitiveness.
Inventor's early stage has been screened and has been obtained two plants of biocontrol trichoderma bacterial strains：Trichoderma harzianum T.harzianum TW21990 and right
Blue Trichoderma spp. T.cyanodichotomus TW21990-1, wherein TW21990 are higher to the suppression ratio of Pythium ultimum, and
TW21990-1 is higher to the rotten mould suppression ratio of melon and fruit, and chlamydospore fermentation level is higher, and can produce blue pigment.Inventor
With above-mentioned two plants of bacterial strains having complementary functions as parent strain, using Protoplast Fusion Technique, selection-breeding obtains efficiently preventing simultaneously
Control the fusant of the mould disease of two kinds of corruption, and the microbial pesticide with chlamydospore as effective ingredient is developed with this, effectively extend system
The shelf life of agent and resistance, lay the foundation for the rotten mould disease of efficient preventing and treating.
Content of the invention
For above-mentioned technical problem, the invention provides a kind of Trichoderma spp. Protoplast fusant and its application, specifically relate to
And a kind of Trichoderma spp. fusant Trichoderma sp.Tpf-2 and this fusant application in the rotten mould damping off preventing and treating of Fructus Lycopersici esculenti.
The deposit number of Trichoderma spp. fusant Trichoderma sp.Tpf-2 of the present invention is CGMCC No.12477, preservation day
Phase is on May 30th, 2016, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Classification And Nomenclature of the present invention is Trichoderma spp. Trichoderma sp..
With to the Trichoderma harzianum T.harzianum TW21990 of Pythium ultimum suppression ratio up to 92.6% and mould to melon and fruit corruption
Suppression ratio up to 83.4% is parent to blue Trichoderma spp. T.cyanodichotomus TW21990-1, is respectively adopted heat-ultraviolet former
Raw plastid inactivation fusion obtains Trichoderma spp. fusant.The mensure fusant rejection ability mould to Pythium ultimum and melon and fruit corruption, protease,
Cellulase, β -1,3- dextranase activity, and liquid fermentation chlamydospore yield level, and through grey Relational Analysis Method
Screening obtains the best fusant Tpf-2 of comprehensive biological and ecological methods to prevent plant disease, pests, and erosion character.
The colony characteristicses of fusant Tpf-2：Aerial hyphae flocculence or graininess, conidium yellow green, yield is less,
Chlamydospore is enriched, and has obvious coconut odors, and light blue pigment is produced at the back side.
Preparation containing fusant Tpf-2 after testing, within 1 year shelf-life, effective chlamydospore survival rate reaches 90%,
Strong stress resistance.Under the conditions of greenhouse and field plot, fusant Tpf-2 can colonize for a long time in Fructus Lycopersici esculenti Rhizosphere Soil, colonizes after inoculation May
Quantity is 1.5 × 105Cfu/g, survival rate reaches 75%, to anti-by the Fructus Lycopersici esculenti damping off that melon and fruit is rotten mould and Pythium ultimum causes
Control effect and respectively reached 86.3% and 85.7%, effect of increasing production has respectively reached 77.3% and 79.9%, effect is significant (p<
0.01).
The present invention compared with prior art has advantages below：
Fusant Tpf-2 synthesis biological and ecological methods to prevent plant disease, pests, and erosion character is excellent, and rotten to melon and fruit mould and Pythium ultimum all has higher inhibitory action, its
Protease, cellulase, β -1,3- dextranase activity is higher, and liquid fermentation chlamydospore yield is big, to Fructus Lycopersici esculenti damping off
Prevention effect is higher, its chlamydospore preparation strong stress resistance, distinctive product blue pigment ability traceable its in plant rhizosphere and
Colonization dynamics in Rhizosphere Soil, are the rotten mould damping off biocontrol bacterial strains of preferable Fructus Lycopersici esculenti.
Brief description
Fig. 1 is the protoplast of trichoderma strain TW21990 and TW21990-1.
The impact that Fig. 2 inactivates to protoplast for Different treatments.
Fig. 3 is the state that two kinds of protoplasts are merging, as indicated by the arrows in the figure.
Fig. 4 is the experiment of the storage life containing chlamydospore preparation.
Fig. 5 is colonization dynamics in Fructus Lycopersici esculenti Rhizosphere Soil for the fusant Tpf-2.
Specific embodiment
To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
Apparent.But embodiment is only exemplary, any restriction is not constituted to the scope of the present invention.Those skilled in the art should
It should be appreciated that, can be to repair to the details of technical solution of the present invention and form under without departing from the spirit and scope of the present invention
Change or replace, but these modifications and replacement each fall within protection scope of the present invention.
Embodiment 1 Trichoderma spp. fusant Tpf-2
The deposit number of Trichoderma spp. fusant tpf-2 of the present invention is CGMCC No.12477, and preservation date is May 30 in 2016
Day, Classification And Nomenclature is Trichoderma spp. Trichoderma sp., and the microbial strains of ginseng evidence are Tpf-2.Depositary institution is China Microbiological
Culture presevation administration committee common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.The present invention by
Trichoderma harzianum T.harzianum TW21990 and blue Trichoderma spp. T.cyanodichotomus TW21990-1 is melted through protoplast
Conjunction forms.
Embodiment 2 protoplast fusion
Experiment parent strain is T.harzianum TW21990 and T.cyanodichotomus TW21990-1.Two kinds
After children's tender mycelia pre-treatment treatment of Trichoderma spp., then the mycelia with lywallzyme Lysing Enzyme cracking TW21990, collapse
Enzyme Driselase cracks the mycelia of TW21990-1, after cracking forms a large amount of protoplast, filters removal with 3 layers of lens paper residual
Broken mycelia, is collected by centrifugation protoplast and is washed with STC, adjusts the protoplasm scale of construction 106Individual/mL.
The biological inoculum deposit number of TW21990 is：CGMCC No.12864, preservation date is：On September 1st, 2016,
Depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is Chaoyang District, Beijing City North Star west
Road 1 institute 3, Classification And Nomenclature is Trichoderma harzianum Trichoderma harzianum.
The biological inoculum deposit number of TW21990-1 is：CGMCC No.12161, preservation date is：2016 2 months 23
Day, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is Chaoyang District, Beijing City north
Occasion West Road 1 institute 3, Classification And Nomenclature is Trichoderma cyanodichotomus.
Result shows, after being processed with pretreating agent beta -mercaptoethanol, the disulfide bond of protein in reducible cell wall, and make thin
Cell wall loosens, and is conducive to the infiltration of lyases, thus improving the formation rate of protoplast.The protoplast that two bacterial strains obtain is general
For circle, not of uniform size, diameter is generally less than 10 μm (Fig. 1).
Respectively two kinds of protoplasts are carried out with heat inactivation and ultraviolet inactivation, inactivation liquid equal-volume mixing, and adds equal-volume
PTC solution promote melt, 35 DEG C of water bath heat preservation 5min, gradient dilution after centrifugation, be mixed in regeneration culture medium, 25 DEG C of constant temperature culture
3d, preserves after the single bacterium colony growing on flat board switching PDA plate.
Protoplast inactivation ratio raises with the prolongation of process time, and wherein TW21990 protoplast ultraviolet processes 15min
Inactivation ratio reaches 55 DEG C of heat treatment 20min inactivation ratios of 100%, TW21990-1 protoplast and reaches 100% (Fig. 2).Inactivation liquid mixes
Merge after adding PTC, parents' protoplast contacts and merges (Fig. 3), according to the damage principle of complementarity of Wright, only double
Parent's fusion just can grow, therefore all in theory parents' fusants growing on regeneration culture medium, on the other hand, due to
TW21990-1 produces blue pigment, and inactivates for heat, only merges and could regenerate with TW21990, therefore should select and produce blue pigment
Regeneration bacterium colony is it is ensured that all parents' fusants of obtaining.This method is obtained 31 plants of fusants, and fusion rate is 10.5%.
Homeo-osmosis agent：0.7mol/L NaCl, 0.2mol/L PBS, pH6.5.
Pretreating agent：0.1% beta -mercaptoethanol.
STC：0.6mol/L Sorbitol, 10mmol/L Tris, 10mmol/L CaC12, pH6.5.
PTC：60%PEG4000,10mmol/L Tris, 10mmol/L CaCl2, pH6.5.
Regeneration culture medium (L)：Add MgCl in PDA20.3mol, inositol 0.3mol, Triton X-100 0.05mL.
Parents' protoplast clump count before clump count/inactivation after fusion rate %=100% × fusion
Embodiment 3 fusant screens
Measure the biological and ecological methods to prevent plant disease, pests, and erosion character of fusant, be mainly included in rotten to melon and fruit mould and Pythium ultimum suppression on PDA plate and make
With；The hydrolysis bandwidth producing on enzyme activity flat board, including β -1,3- glucanase, cellulase and protease；Liquid fermentation
Chlamydospore yield.Carry out overall merit using the fusant that grey Relational Analysis Method obtains to primary dcreening operation, big according to the degree of association
The excellent fusant of little determination.
Suppression ratio %=100% × (comparison pathogen growth radius-process pathogen growth radius)/comparison pathogen growth radius
50 × trace element (L)：FeS04·7H20 0.25g, ZnSO4·2H2O 0.07g, CoCl2·6H2O 0.1g,
MnSO4·H2O 0.085g.
Beta-1,3-glucanase culture medium (L)：Glucose 20g, yeast extract 0.67g, K2HPO415g, (NH4)2S04
5g, CaCl20.6g, MgCl20.6g, 50 × trace element 20mL, peptone 2g, aniline orchid 60mg, pachyman 4g, Poria
Powder 3g, Triton X-100 0.05mL, agar 15g.
Protease culture medium (L)：Glucose 0.5g, gelatin 10g, Triton X-100 0.05mL, agar 15g.
Cellulase culture medium (L)：Glucose 0.5g, KH2PO415g, (NH4)2SO45g, CaCl20.6g, MgSO4·
7H2O 0.6g, FeSO4·7H2O 0.005g, MnSO4·H2O 0.0016g, ZnSO4·7H2O 0.0014g, CoCl2·6H2O
0.0037g, CMCNa 10g, Triton X-100 0.05mL, agar 15g.
Fermentation medium (L)：Glucose 0.5g, Semen Maydis powder 4g, yeast extract 1g, (NH4)2SO42g, KH2PO40.1g,
MgSO40.05g, pH5.0.
Result shows, according to flat board bacteriostasis, from 31 plants of fusants, primary dcreening operation obtains 9 plants of fusants, this 9 plants of fusants
And the biological and ecological methods to prevent plant disease, pests, and erosion character measurement result of parent strain is shown in Table 1, and determine the ideal value composition reference strain of biocontrol trichoderma bacterial strain.
The biological and ecological methods to prevent plant disease, pests, and erosion character measurement result of 19 plants of primary dcreening operation fusants of table
Can be seen that (table 2) after grey relational grade analysis, the grey relational grade of 9 plants of fusants of primary dcreening operation and reference strain
Both greater than parent strain, the wherein grey relational grade of fusant Tpf-2 are maximum, be 0.9198, the comprehensive of 9 plants of fusants of primary dcreening operation is described
The anti-character of symphysis is above parent strain, and Tpf-2 effect is especially notable.
Table 2 is for trying grey relational grade and the sequence of fusant
The suppression of and Pythium ultimum rotten to melon and fruit mould by the grey relational grade analysis excellent fusant Tpf-2 that obtains of screening
Ability, chlamydospore fermentation level, and protease, cellulase and β -1,3- glucanase hydrolysing activity is all higher.Particularly
Its β -1,3- glucanase hydrolysing activity is higher, and this hydrolytic enzyme is to prevent and treat one of crucial enzyme system of rotten mould disease, and two parents are simultaneously
No this enzyme hydrolysiss activity.
The morphological characteristic of embodiment 4 fusant Tpf-2
Fusant Tpf-2 and parent strain 25 DEG C of culture 7d on PDA plate, colonial morphology is different from two parents,
With parent TW21990-1 more closely, predominantly aerial hyphae flocculence or graininess, conidium yellow green, yield is less,
Chlamydospore is enriched, and has obvious coconut odors, and light blue pigment is produced at the back side.
The chlamydospore preparation containing fusant Tpf-2 for the embodiment 5
Fusant Tpf-2,25 DEG C, 120r/min shaken cultivation 5d are cultivated using fermentation medium, is collected using membrane filtration
Chlamydospore, in following ratio preparation of preparation (wettable powder), wherein turfy soil is carrier, and chlamydospore is that effective viable bacteria becomes
Point, other is auxiliary agent.
Preparation is put Room-temperature seal and is preserved, periodically sampling detection chlamydospore content alive.
Result is shown in Fig. 4, and chlamydospore preparation initial content is 200,000,000/g, and after room temperature preservation December, chlamydospore content is
1.8 hundred million/g, preparation effective Conidia persistence within 1 year shelf-life reaches 90%, the Trichoderma spp. bacteria preparation discussion solicited opinions
Specify in original text：Preparation effective Conidia persistence within 1 year shelf-life reaches 80%, therefore this preparation has met and exceeded above-mentioned finger
Mark.
The greenhouse of embodiment 6 fusant Tpf-2 and field plot trial
Fusant Tpf-2 was transferred for 10 generations, after detecting its stable heredity, carries out greenhouse and field plot trial, greenhouse tries
Testing with pathogenic fungi is the melon and fruit mould and Pythium ultimum of corruption, and trial crops are Fructus Lycopersici esculenti, and comparison medicament is metalaxyl, and test sets 3 weights
Multiple, 4 weeks investigation planting percents of emerging, calculate prevention effect.
Prevention effect %=100% × (processing planting percent-comparison planting percent)/(100- compares planting percent)
Fusant Tpf-2 has preferable prevention effect (table 3) under greenhouse experiment to Fructus Lycopersici esculenti damping off, with parent strain
And between comparison medicament metalaxyl, all there is significant difference (p<0.01), to the preventing and treating by the rotten mould Fructus Lycopersici esculenti damping off causing of melon and fruit
Effect has reached 86.3%, has been respectively increased 33.2% and 18.1% compared with parent strain TW21990 and TW21990-1；To by end
The prevention effect of the extremely rotten mould Fructus Lycopersici esculenti damping off causing has reached 85.7%, compared with parent strain TW21990 and TW21990-1 respectively
Improve 15.4% and 28.7%.
The greenhouse prevention effect to Fructus Lycopersici esculenti damping off for the table 3 fusant Tpf-2
Note：Data adopts Duncan ' the s method in SPSS 19.0 statistical software to be analyzed, and result is 3 repetitions
Mean+SD, represents there is significant difference in 0.01 level between each process of same column with different capitalizations.
Greenhouse test statistics terminates, and tomato seedling is transplanted to field plot, and with fusant and parent strain to children
Seedling root carries out 2 times pouring applies preparation process, plot area 16m2, often process and set 3 repetitions, random distribution, period Routine Management,
In periodically sampling detection Rhizosphere Soil, the colonization dynamics of fusant Tpf-2, produce the energy of blue pigment because fusant Tpf-2 is distinctive
Power, therefore differentiate labelling as reclaiming.Count yield after Fructus Lycopersici esculenti harvests, calculate effect of increasing production.
Effect of increasing production %=100% × (treatment group yield-matched group yield)/matched group yield
Fusant Tpf-2 successfully can colonize (Fig. 5) in Fructus Lycopersici esculenti Rhizosphere Soil, and after colonizing 0.5 month, quantity is in rising trend,
Illustrate that Tpf-2 can breed in soil, after colonizing January, quantity maintains initial inoculation level, and quantity slowly declines thereafter, to Fructus Lycopersici esculenti
Harvest (after May), colonizing quantity after inoculation May is 1.5 × 105Cfu/g, colonize that quantity is initial inoculum 75%, explanation
The chlamydospore of fusant Tpf-2 effectively can colonize in Fructus Lycopersici esculenti Rhizosphere Soil for a long time, is that disease-preventing and yield-increasing is laid a good foundation.
Fusant Tpf-2, to tomato production promoting effect is significant (table 4), all exists aobvious and between two parents and comparison medicament metalaxyl
Write sex differernce (p<0.01), wherein reached 77.3% to by the effect of increasing production of the rotten mould Fructus Lycopersici esculenti damping off causing of melon and fruit, to by
The effect of increasing production of the Fructus Lycopersici esculenti damping off that Pythium ultimum causes has reached 79.9%.
The field plot effect of increasing production to Fructus Lycopersici esculenti damping off for the table 4 fusant Tpf-2
Note：Data adopts Duncan ' the s method in SPSS 19.0 statistical software to be analyzed, and result is 3 repetitions
Mean+SD, represents there is significant difference in 0.01 level between each process of same column with different capitalizations.
Claims (9)
1. a kind of Trichoderma spp. Protoplast fusant it is characterized in that, Trichoderma spp. Protoplast fusant be Trichoderma spp. fusant Tpf-
2, its deposit number is CGMCC No.12477, and preservation date is on May 30th, 2016, and depositary institution is Chinese microorganism strain
Preservation administration committee common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Classification And Nomenclature is wood
Mould Trichoderma sp..
2. the chlamydospore that Trichoderma spp. Protoplast fusant fermentation as claimed in claim 1 obtains.
3. contain Trichoderma spp. Protoplast fusant as claimed in claim 1 or contain chlamydospore as claimed in claim 2
Preparation.
4. Trichoderma spp. Protoplast fusant as claimed in claim 1 preventing and treating melon and fruit is rotten mould and Pythium ultimum in terms of application；
Preferably, in preventing and treating by the application in terms of the rotten mould Fructus Lycopersici esculenti damping off causing with Pythium ultimum of melon and fruit.
5. the preparation containing Trichoderma spp. Protoplast fusant as claimed in claim 1 is in the rotten mould and Pythium ultimum side of preventing and treating melon and fruit
The application in face；Preferably, in preventing and treating by the application in terms of the rotten mould Fructus Lycopersici esculenti damping off causing with Pythium ultimum of melon and fruit.
6. the chlamydospore that Trichoderma spp. Protoplast fusant fermentation as claimed in claim 2 obtains is rotten mould and whole in preventing and treating melon and fruit
The application of extremely rotten mould aspect；Preferably, in preventing and treating by the application in terms of the rotten mould Fructus Lycopersici esculenti damping off causing with Pythium ultimum of melon and fruit.
7. the chlamydosporic preparation containing Trichoderma spp. Protoplast fusant fermentation acquisition as claimed in claim 2 is in preventing and treating melon
The rotten mould application with Pythium ultimum aspect of fruit；Preferably, in preventing and treating by the rotten mould Fructus Lycopersici esculenti damping off causing with Pythium ultimum of melon and fruit
The application of aspect.
8. preparation according to claim 3 is it is characterised in that preparation formulation is wettable powder, by following parts by weights
The group of number meter is grouped into：
9. prepare the method for Trichoderma spp. Protoplast fusant as claimed in claim 1 it is characterised in that protoplast fusion walks
In rapid, 15min is processed to parent's TW21990 protoplast ultraviolet, to 55 DEG C of heat treatments of parent's TW21990-1 protoplast
20min.
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| CN111269844A (en) * | 2020-04-02 | 2020-06-12 | 山东省科学院生态研究所 | Trichoderma harzianum TW21990 for preventing and treating leek gray mold and application thereof |
| CN111455015A (en) * | 2020-04-02 | 2020-07-28 | 山东省科学院生态研究所 | Rapid screening method of continuous cropping obstacle factor strains for degrading phenolic acid substances |
| CN111269844B (en) * | 2020-04-02 | 2021-05-25 | 山东省科学院生态研究所 | Trichoderma harzianum TW21990 for preventing and treating leek gray mold and application thereof |
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