CN101492646A - Trichoderma viride engineering bacterium and uses thereof - Google Patents
Trichoderma viride engineering bacterium and uses thereof Download PDFInfo
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Abstract
The invention discloses Trichoderma viride engineering bacteria, which comprises endochitinase genes, was named as green Trichoderma viride R65 and was preserved in 'CCTCC' on December 29, 2008. The preserving number is CCTCC NO: M 208265. The invention also discloses the applications of the strains in controlling vegetables. The highest vigor of the chitinase of the strains can achieve 77.4U/ml, which is 3.67 times of the strains of an original host. The strains have stronger antagonism on a plurality of plant pathogenic fungi like cucumber gray mold, Aspergillus niger, rhizoctonia solani kuhn, wheat red mould, cotton Phytophthora, and the like, and is an engineering bacteria which have more application values in controlling vegetable.
Description
Technical field
The invention belongs to microbial project and biological control technical field; Specifically, the present invention relates to a strain and cross the engineering bacteria trichoderma viride engineering bacterium of expressing endo-chitinase genes, also relate to the application of this bacterial strain in controlling vegetables.
Background technology
Wood mould (Trichoderma spp.) belongs to Deuteromycotina, hyphomycetes, and clump falx order, clump falx section is the widely distributed soil inhabitant of a class, is common in plant residue and the animal excrement.Except that being the weak pathogenic bacteria individually, great majority have antagonistic action to phytopathogen.Because mould diversity and the parasitic broad spectrum that has extensive adaptability, produces antagonist of wood, thereby be widely studied as the antagonism bacterium of biocontrol of plant disease.
Trichoderma comprises competition effect, hyperparasitism and antibiosis etc. to the biological and ecological methods to prevent plant disease, pests, and erosion mechanism of action of phytopathogen.The hyperparasitism of Trichoderma is the main mechanism of its antagonism pathogenic fungi, it has comprised invasion and attack, identification, contact to pathogenic bacteria, twine, penetrate and complex process (the Elad Y of a series of consecutive steps such as parasitism, Barak R, Chet I.Parasitismof Sclerotium rolfsii by Trichoderma harzianum[J] .Soil Biol Biochem, 1984,16:381-386).Studies show that, after Trichoderma and the identification of host fungi, the Trichoderma silk twines growth along parallel growth of host mycelia and spirrillum, and generation appressorium shape branch is adsorbed on the host mycelia, by secretion extracellular enzyme dissolved cell wall, penetrate the host mycelia, draw nutrition (Elad Y, Chet I, Boyle P, et al.Parasitism of Trichoderma spp.on Rhizoctoniasolani and Sclerotium rolfsii scanning electron microscopy and fiuorescence microscopy[J] .Phytopathology, 1983,73:85-88).Most scholars think that the antagonism Trichoderma has produced the lytic enzyme of a series of degraded pathogenic bacteria cell wallss in parasitic processes, as chitinase, cellulase, zytase, dextranase and proteolytic enzyme etc.Studies show that, 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 β-1,3-dextranase (Lorito M, Hannan GE, Hayes CK, et al.Chitinolytic enzymes produced by Trichodermaharzianum:antifungal activity of purified endochitinase and chitobinase[J] .Phytopathology, 1993,83:302-307; Sela-Buurlage MB, Ponstein AS et al.Only specific tobacco-chitinase and β-1,3-glucanase exhibit antifungal activity[J] .Plant Physiology, 1993,101:857-863).
Viride (Trichoderma viride) is the important Trichoderma of a class, has widely distributed, growth fast and characteristics such as the lasting period is long, be widely used in plant soil-borne diseases biological control (Howell C R.Mechanisms employed by Trichoderma species in the biological control of plant diseases:The history and evolution of current concepts[J] .Plant Disease, 2002,87, (1): 4-10).But present employed viride biological prevention and control agent still exists the validity of further raising biological and ecological methods to prevent plant disease, pests, and erosion effect and the problem of stability.Along with being gradually improved of genetic engineering technique, the gene that the clone transforms the antagonism pathogenic fungi makes up the emphasis that the engineering strain with high antagonism usefulness has become biological and ecological methods to prevent plant disease, pests, and erosion work in Trichoderma.Because chitinase plays an important role in Biocontrol Mechanism, its correlative coding gene is being subjected to extensive concern aspect the transformation of biological and ecological methods to prevent plant disease, pests, and erosion engineering bacteria.Yet, at present domestic yet there are no by crossing the work that endo-chitinase genes makes up the mould engineering bacteria of efficient green wood of expressing.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provides a strain and crosses viride engineering bacteria and the application of this bacterial strain in controlling vegetables of expressing endochitinase.Utilize the spore liquid preparation of this engineering strain can effectively improve the resistibility of vegetables such as cucumber, tomato, and improve vegetable crop simultaneously fungal disease.
Viride engineering bacteria of the present invention, contained expression inscribe chitin gene, this bacterium called after viride (Trichoderma viride) R65 (ech42), and be preserved in 2008 months 29 days on the 12nd that " Chinese typical culture collection " center ", preserving number is CCTCC N0:M 208265.
The morphological specificity of above-mentioned viride engineering bacteria is: on the PDA substratum, its bacterium colony is the velvet-like of colourless or light color, and later stage bacterium colony surface begins to occur green, the visible concentricity in gonimoblast bundle district; Mycelia is separated, and branch is complicated; To giving birth to or the alternate branch, can continue branch again on the branch on the mycelia brachyplast, branch is acute angle or intimate right angle, Shu Sheng, to giving birth to or the Dan Shengmo branch; Conidium is born in succession by final terminal branch, by mucus their conglomerates or subglobose spore submanifold; Conidium is subsphaeroidal, ellipse or round shape, transparent or bright green.
The bacterium colony of above-mentioned viride engineering bacteria, mycelia and spore shape are seen Fig. 1,2.
The construction process of above-mentioned viride engineering bacteria: according to conventional filamentous fungus gene transformation method, the fungi plasmid p3SR2 that endo-chitinase genes is crossed expression vector pG-ech42 and carried the acetamidase expression casette obtains the possible transformant of 26 strains with 3: 1 mixed cotransformation original strain viride (T.viride) W512 protoplastiss on the substratum that with the ethanamide is sole carbon source and nitrogenous source.Extract the transformant chromosomal DNA, checking obtains a strain transformant through PCR, with its called after viride (Trichoderma viride) R65.The checking transformant the results are shown in Figure 3.
It is the highest to produce chitinase activity during above-mentioned viride engineering bacteria liquid fermenting 48hr, (enzyme work is with pH5.8 for 77.4U/ml, per hour decompose the N-acetylglucosamine that tobacco brown spot pathogen discharges 1ug when temperature is 37 ℃ and be defined as an enzyme activity unit (U)), be 3.67 times of original strain viride (T.viride) W512; This project bacterial strain shows in flat board face-off phytopathogens such as cucumber grey mold, aspergillus niger, dry thread Pyrenomycetes, gibberella saubinetii, cotton epidemic disease are mould than the high restraining effect of original strain viride (T.viride) W512.
The application of viride engineering bacteria of the present invention in controlling vegetables.
Wherein: described viride engineering bacteria is implemented with the biological prevention and control agent form; Wherein said biological prevention and control agent prepares with following method: R65CCTCC N0:M 208265 is inoculated in the PDA solid medium with viride (Trichoderma viride), cultivated 4～5 days for 28～30 ℃, with sterilized water or cold water spore is washed, collecting spore and regulating its concentration is 1～5 * 10
7Individual/ml, obtain the spore suspension preparation.
Above-mentioned PDA solid medium preparation: potato 200g, boiling tap water 30min, 6 layers of filtered through gauze add glucose 20g in filtrate, agar 17g, moisturizing is to 1000ml.
Described viride engineering bacteria is implemented the method for controlling vegetables: under 20～30 ℃ of envrionment temperatures, be 1～5 * 10 with concentration
7The spore liquid of individual/ml is irritated root to the potted plant vegetable seedling that was unearthed 10 ± 1 days, handles 1～2 time, every 10 days once (each time of application is afternoon 3～4 points); Or be 1～5 * 10 with concentration
7The spore liquid of individual/ml sprays the vegetables of booth field planting, handles 3～6 times, every 10 days once (each time of application is afternoon 3～4 points).
In the above-mentioned application: damping-off preferably adopts root irrigation; Powdery Mildew and gray mold preferably adopt and spray.
In the above-mentioned application: described vegetables are cucumber or tomato preferably.
Viride engineering bacteria of the present invention can be prevented and treated effectively and various vegetables such as tomato, cucumber are caused the pathogenic fungi of disease, and vegetable crop is increased to some extent.
For example: after using above engineering strain preparation, potted plant cucumber does not have the state of an illness to take place, and this biocontrol effect with respect to original strain viride (T.viride) preparation 70.13% is significantly improved; For plastic tent cucumber, the said preparation biocontrol effect reaches 92%, has improved 18% than original strain viride (T.viride) preparation, uses simultaneously that cucumber yield improves 21.9% behind the said preparation; For tomato in plastic greenhouse, the biocontrol effect of using this project bacterial strain preparation reaches 95%, has improved 23% than original strain viride (T.viride) preparation, makes tomato yield improve 21.9% simultaneously.
Viride of the present invention (Trichoderma viride) R65 CCTCC N0:M 208265 has very high chitinase activity, and producing chitinase activity during liquid fermenting 48hr is 77.4U/ml, is 3.67 times of original strain (21.1U/ml); Multiple pathogenic fungi all has very strong antagonistic action to this bacterial strain to cucumber grey mold, aspergillus niger, dry thread Pyrenomycetes, gibberella saubinetii, cotton epidemic disease be mould etc.The biological prevention and control agent preventive effect made of bacterial strain is than original strain viride (T.viride) W512 height thus, and can increase substantially the output of the vegetables of trying simultaneously, and this project bacterium is the biocontrol strain that a strain has using value very much.
Description of drawings
Fig. 1. viride (Trichoderma viride) R65 CCTCC N0:M208265 colonial morphology
Fig. 2. viride (Trichoderma viride) R65 CCTCC N0:M208265 mycelia and spore shape
Fig. 3. detected expression vector pG-ech42 transformant PCR product electrophorogram
Wherein: M, λ DNA/Pst; 1, the pG-ech42 positive control; 2, R65 transformant chromosomal DNA; 3, the W512 chromosomal DNA.
Fig. 4. endo-chitinase genes is crossed expression vector pG-ech42 physical map
Specific embodiment
The structure of embodiment 1. engineering strain virides (Trichoderma viride) R65CCTCC N0:M 208265
Endo-chitinase genes is crossed expression vector pG-ech42 and is made up (Wang Xiaodong by State Key Laboratory for Microbial Technology (Jinan) Wang Xiaodong, antifungal protein and the research of chitinase gene cloning and expression, the master of Shandong University Diplomarbeit, 2006), this carrier endochitinase ech42 upstream region of gene contains the gpd gene strong promoter that derives from Aspergillus nidulans, endo-chitinase genes is crossed the physical map of expression vector pG-ech42 and is seen Fig. 4, and its building process is as follows:
With pAN7-1 is the gpd promotor of template about primer p1/p2PCR amplification 1.1k, and primer p1/p2 and PCR process are as follows:
p1：5′-GCGC
GAATTCAGACCTAATACAGCCCCTAC-3′
EcoRI
p2：5′-GCGC
GGTACCTGTCTGCTCAAGCGGGGTAG-3′
KpnI
PCR condition: 95 ℃ of 5min, 94 ℃ of 0.5min → 52 ℃ 0.5min → 72 ℃ of 1.5min (35 circulations), 72 ℃ of 10min.Polysaccharase is that general T aq and high-fidelity pfu mix, and template is plasmid pAN7-1.
Behind the purified test kit purifying of PCR product, with EcoRI and KpnI double digestion, the purification kit purifying obtains gpd promotor segment; Behind pUC19 plasmid usefulness EcoRI and KpnI double digestion, use T behind the glue recovery purification kit purifying with after the gpd promotor that obtains is above mixed
4Ligase enzyme connects, and obtains recombinant plasmid pPgpd, transformed into escherichia coli DH5 α.Behind pAN7-1 plasmid usefulness BamHI and HindIII double digestion, reclaim the TtrpC terminator that purification kit reclaims the 0.8kb that scales off with glue.The pPgpd plasmid with BamHI and HindIII double digestion, is connected with the TtrpC terminator that obtains above behind the glue recovery purification kit purifying, and transformed into escherichia coli DH5 α obtains recombinant plasmid pG.
Pcr amplification W512 endochitinase ech42 gene primer sequence is as follows:
Ech-s：5′-TAG
GGTACCATGTTGGGCTTCCTCGGA-3′
KpnI
Ech-a：5′-CTC
GGATCCCTAGTTGAGACCGCTTCG-3′
BamHI
Template is plasmid pMDECH, and the PCR condition is: 95 ℃ of 5min, 94 ℃ of 0.5min → 58 ℃ 0.5min → 72 ℃ of 1.5min (35 circulations), 72 ℃ of 10min.Polysaccharase is that general T aq and high-fidelity pfu mix.The endochitinase ech42 mrna length that amplification obtains should be about 1500bp.Behind the purified test kit purifying of PCR product, with BamHI and KpnI double digestion, purification kit purifying, with the pG T after enzyme is cut equally
4Ligase enzyme connects, and transformed into escherichia coli DH5 α obtains containing the bacterial strain of pG-ech plasmid.
Original host strain is viride (T.viride) W512, and this bacterial strain has identical feature with green tricoderma LTR-2-2 bacterial strain in gene expression characteristics and the inhibition to various pathogenic bacterias.
Endo-chitinase genes is crossed expression vector pG-ech42 and carried the fungi plasmid p3SR2 of acetamidase expression casette with 3: 1 mixed cotransformation original strain viride W512 (T.viride W512) protoplastiss, the enterprising row filter of substratum with the ethanamide being sole carbon source and nitrogenous source tentatively obtains the possible transformant of 26 strains.Detailed process is as follows:
The preparation of protoplastis:
1. viride (T.viride) W512 spore suspension dilution coating ethanamide fungi transforms culture medium flat plate, selects and utilizes the bacterial strain a little less than the ability to be starting strain to ethanamide.The switching potato slope is given birth to spore, collects spore.
2. place a glassine paper on the potato culture medium flat plate, coating starting strain spore suspension is cultivated 20hr for 28 ℃ on it.
3. the mycelia on the glassine paper in the flat board is shifted, is suspended in 1.2mol/L sorbyl alcohol-10mmol/L potassium phosphate solution (pH5.6 contains 5mg/ml-10mg/ml Lysing Enzymes), 28 ℃ of temperature are bathed 1.5～2hr.Microscopy frequently in this process, the generation situation of observing protoplastis.
4. filter by the G3 sinter funnel, protoplastis and the mycelium that is not digested are separated, and washed 2 times with 1.2mol/L sorbyl alcohol-10mmol/LTrisHCl (pH7.5).
5. the centrifugal 15min of 4000rpm abandons supernatant, collects protoplastis, and washs 2 times with 1mol/L sorbyl alcohol-10mmol/LTrisHCl (pH7.5).Be resuspended in 1mol/L sorbyl alcohol-10mmol/LCaCl at last
2Among-10mmol/L the TrisHCl (pH7.5), making protoplastis concentration is 5 * 10
6-5 * 10
7/ ml.
The conversion of protoplastis:
The plasmid p3SR2 and the purpose of carrying from A.nidulans coding acetamidase amdS gene behind the purifying are transformed each 2～5 μ g (maximum 20 μ l of plasmid pG-ech42, both mass ratios 1: 3) mix with viride (T.viride) W512 protoplastis (100 μ l), add 50 μ l 25%PEG6000-50mmol/L CaCl
2-10mmol/LTrisHCl (pH7.5), ice bath 20min, the back adds 1ml 60%PEG4000-50mmol/L CaCl
2-10mmol/L TrisHCl (pH7.5) (adding volume is plasmic 10 times), room temperature is placed 5min.Add 2ml 1mol/L sorbyl alcohol-10mmol/LCaCl at last
2-10mmol/L TrisHCl (pH7.5), mixing.
Regeneration and selection: an amount of (100 μ l～200 μ l) above-mentioned conversion fluid is coated on the ethanamide fungi conversion culture medium flat plate, cultivated 3～4d for 28 ℃, select the bigger transformant of the very fast bacterium colony of growth on ethanamide fungi conversion substratum.While carries out in the same way for the protoplastis that does not add the purpose plasmid, is coated on by identical amount not contain and contain on the sorbyl alcohol minimum medium that ethanamide is sole carbon source and nitrogenous source respectively as protoplast regeneration situation and negative control.
The used ethanamide fungi of above conversion test transforms substratum: ethanamide 10mmol/L, KH
2PO
415g/L, MgSO
40.6g/L, CaCl
20.6g/L, FeSO
47H
2O 0.005g/L, MnSO
4H
2O 0.0016g/L, ZnSO
47H
2O 0.0014g/L, CoCl
20.002g/L, Triton X-1001ml/L, agar 20g/L, pH5.5.
The transformant of selecting is transferred in the potato medium slant, cultivate about 3～5d for 30 ℃.Well can not consider behind switching 4～5d if give birth to spore, choose some mycelia from potato slope and continue the living spore of switching potato slope.After treating that spore grows up to green, with spore under the aseptic water washing, collect spore and add 80% glycerine equal-volume and mix, a part is put-20 ℃ and is stored for future use, and whether another part is put on the ethanamide fungi conversion culture medium flat plate and cultivated, observe transformant and grow.If the growth this transformant would be preserved as positive transformant.
Extract the transformant chromosomal DNA, carry out PCR checking (the primer is to amplifying the 2.6kb dna segment that comprises gpdA promotor and ech42 structure gene, and primer and PCR program are as follows).
P3：5’-GCGCGAATTCAGACCTAATACAGCCCCTAC-3′
P4：5’-CTCGGATCCCTAGTTGAGACCGCTTCG-3′
The PCR condition is: 95 ℃ of 5min, and 94 ℃ of 0.5min → 48 ℃ 0.5min → 72 ℃ of 2.5min (35 circulations), 72 ℃ of 10min, amplification gpd promotor and chitinase gene total length, product is long to be 2.6kb.
Checking obtains a strain transformant, this bacterium called after viride (Trichoderma viride) R65 through PCR.This bacterial strain has been preserved on December 29th, 2008 that " Chinese typical culture collection " center ", preserving number is CCTCC N0:M 208265.
] embodiment 2. virides (Trichoderma viride) R65CCTCC N0:M 208265 morphologic observations
R65CCTCC N0:M 208265 is inoculated into the PDA substratum with viride (Trichoderma viride), cultivates 3～4 days for 30 ℃, observes colony characteristics such as Fig. 1; The cotton blue dyeing of mycelia is observed, and uses the NIKONALPHAPHOT-2 system to take a picture mycelia and spore shape such as Fig. 2.
On the PDA substratum, its bacterium colony is the velvet-like of colourless or light color, and later stage bacterium colony surface begins to occur green, the visible concentricity in gonimoblast bundle district; Mycelia is separated, and branch is complicated.To giving birth to or the alternate branch, can continue branch again on the branch on the mycelia brachyplast, branch is acute angle or intimate right angle, Shu Sheng, to giving birth to or the Dan Shengmo branch; Conidium is born in succession by final terminal branch, by mucus their conglomerates or subglobose spore submanifold.Conidium is subsphaeroidal, ellipse or round shape, transparent or bright green.
With reference to Tan Jianxin (Tan Jianxin, the research of Bt cry gene clone and Bacillus circulans chitinase. the doctor of graduate school of Chinese Academy of Agricultural Sciences academic dissertation, 2001) the chitinase activity measuring method carries out chitinase activity to viride R65CCTCC M208265 and measures.Concrete grammar is as follows:
Helicase liquid preparation: take by weighing helicase dry powder 1g, EDTA (0.5mol/L) 0.2mL, KCl (1mol/L) 1mL places mixing in the volumetric flask, add distilled water to the 100ml dissolving fully, get final product 1% helicase, cryopreservation.
N-acetylglucosamine standard curve making: N-acetylglucosamine (GlcNAc) mother liquor of configuration 100ug/ml is diluted to this mother liquor respectively different concns (0,20,40,60,80,100ug/ml) again; Get several clean test tubes then respectively, add the N-acetylglucosamine 0.4ml of different concns respectively, add the saturated borax soln of 0.2ml, boiling water bath 7min, add 2ml Glacial acetic acid and 1ml1% paradimethy laminobenzaldehyde solution after the cooling, behind 37 ℃ of insulation 15min, measure OD value under the 585nm; With the OD value is ordinate zou, and N-acetylglucosamine concentration is X-coordinate drawing standard curve.
Enzyme activity determination: get crude enzyme liquid 0.4mL, add acetate buffer 0.4mL (0.1mol/L, PH 5.8), 0.4mL tobacco brown spot pathogen (0.1%), 37 ℃ are reacted 6h down, centrifugal (10000rpm, 10min); Get the 0.4mL supernatant liquor, add 1% helicase of 0.1mL, 37 ℃ of reaction 1h down are contrast with the reaction solution that comprises substrate and enzyme (heated and inactivated) of same processing.Wherein the N-acetylglucosamine Determination on content of Chan Shenging obtains the concrete N-acetylglucosamine content in OD value back and is read by N-acetylglucosamine typical curve with measuring method in the standard curve making process.
Chitinase activity definition: in above-mentioned enzyme reaction system, enzyme work is defined as 1 enzyme activity unit (U) with the N-acetylglucosamine that per hour decomposes tobacco brown spot pathogen and discharge 1ug.
Viride (Trichoderma viride) R65CCTCC N0:M 208265 and contrast original strain viride (T.viride) W512 are carried out chitinase activity to be measured when showing 48hr, it is maximum that the enzyme activity of viride (Trichoderma viride) R65CCTCC N0:M 208265 and original strain viride (T.viride) W512 reaches, be respectively 77.4U/ml and 21.1U/ml, the chitinase activity of engineering strain has improved 2.67 times than original strain.This demonstration transforms former host strain again with endo-chitinase genes, and the endo-chitinase genes that changes over to inserts on the viride karyomit(e) and successful expression.Since the endo-chitinase genes copy number increase enhancing with genetic expression, transformant excretory chitinase activity is greatly improved.
Above-mentioned used liquid fermenting produces the enzyme substratum: glucose 20g/L, (NH
4)
2SO
45g/L, KH
2PO
415g/L, MgSO
40.6g/L, CaCl
20.6g/L, FeSO
47H
2O 0.005g/L, MnSO
4H
2O 0.0016g/L, ZnSO
47H
2O 0.0014g/L, CoCl
20.002g/L (pH5.5).
Above-mentioned crude enzyme liquid is 4 ℃ of fermented liquids, gets supernatant behind the centrifugal 4min of 6000rpm and obtains.
Embodiment 4. dull and stereotyped face-off tests
Method (Bell K with reference to people such as Bell K, Wells HD, Markham CR.Invitroantagonism ofTrichoderma species against fungal pathogens[J] .Phytopath.1982,72:379-382), both sides are at a distance of difference dibbling viride (Trichoderma viride) R65CCTCC N0:M 208265 (or original strain viride (T.viride) W512) of 5cm place and cucumber grey mold on the PDA flat board, aspergillus niger, dry thread Pyrenomycetes, gibberella saubinetii, the mould grade of cotton epidemic disease plant pathogenic fungi, cultivate down, observe the growing state of dull and stereotyped last two kinds of bacterium for 28～30 ℃.Compare with independent inoculation viride (Trichoderma viride) the R65 CCTCC N0:M 208265 (or original strain viride (T.viride) W512) and the flat board of various pathogenic fungies.Day by day observe the growth of bacterium colony and the restraining effect of engineering strain, observe 5～7d continuously, every processing repeats 3 times.The relative original strain viride of inhibition effect (T.viride) W512 of phytopathogens such as the result shows, 208265 pairs of cucumber grey mold of the engineering strain viride of overexpression endo-chitinase genes (Trichoderma viride) R65 CCTCC N0:M, aspergillus niger, dry thread Pyrenomycetes, gibberella saubinetii, cotton epidemic disease are mould has raising in various degree.
The making of embodiment 5. virides (Trichoderma viride) R65 CCTCC N0:M 208265 biological prevention and control agents
Viride (Trichoderma viride) R65 CCTCC N0:M 208265 is inoculated in the PDA solid medium, cultivates after 4～5 days for 28～30 ℃, spore being washed and regulates its spore concentration with sterilized water or cold water is 1～5 * 10
7Individual/ml makes the spore suspension preparation.
Above-mentioned PDA solid medium preparation: potato 200g, boiling tap water 30min, 6 layers of filtered through gauze add glucose 20g in filtrate, agar 17g, moisturizing is to 1000ml.
The application of embodiment 6. virides (Trichoderma viride) R65 CCTCC N0:M 208265 on potted plant cucumber
Carrying out two batches of pot experiments altogether, is " helping excellent No. 10 " for the examination cucumber variety.The first phase pot experiment; get the soil of not planting vegetables in recent years; smash to pieces than large clod; 11.5 centimetres of internal diameters of dress, high 10 centimetres plastic tub; water the water of revealing the exact details, live 3 Semen Cucumidis sativi of every basin after 4 hours (through 26 ± 2 ℃ of vernalization to showing money or valuables one carries unintentionally) are covered thin dry ground 1-1.5cm; inquire for sunlight after coming up, every basin keeps 2 seedlings behind the tidy seedlings output.Emerging back 10 ± 1 days, be 10 with concentration
7The engineering strain of individual/ml and original strain spore suspension preparation are irritated root respectively, and each bacterial strain is all established 5 times of dilutions and do not diluted (directly irritating root 30ml stoste) two processing, repeat 5 times, and be contrast to water clear water.Test and investigate indexs such as disease index and biocontrol effect latter stage.The second phase pot experiment is carried out after two months in that first phase is potted plant, and method is the same, first phase from late June to by the end of July, second phase from late September is to by the end of October.
The potted plant result of first phase shows that plant is encroached on by pathogenic bacteria all; Second phase, the result of pot experiment was as shown in table 1.Engineering strain viride (Trichoderma viride) R65 CCTCC N0:M 208265 preparation biocontrol effects have increased significantly than original strain viride (T.viride) W512 as shown in Table 1, average biocontrol effect improves 29.87%, and plant is not found the state of an illness after using engineering strain.
The second stage of pot experiment disease index of table 1 and biocontrol effect table
The application of embodiment 7. virides (Trichoderma viride) R65 CCTCC N0:M 208265 on plastic tent cucumber and tomato
Tomato in plastic greenhouse single factor experiment: arrange single factor experiment, investigate the biocontrol effect of the spore suspension preparation of engineering strain viride (Trichodermaviride) R65 CCTCC N0:M 208265 and viride (T.viride) W512 comprehensively, investigate plant height, stem raw data simultaneously, analyze the growth-promoting functions of engineering strain under the state of land for growing field crops.Experimental field be damp cinnamon soil, soil organic matter content 1.18%, quick-acting nitrogen content 135ppm, available phosphorus contents 35ppm, available potassium 108ppm, continuous 3 years planting vegetables before the test; This canopy is in the phase second largest stubble of knot tomato beginning in field planting on September 15 when arranging test, be " the super hat of Jinpeng " for the examination tomato variety; Per 10 ± 1 days, spray 10 at fine day 3-4 point in afternoon
7The engineering strain of individual/ml spore concentration and original strain spore suspension preparation connect and spray 4 times; Sub-district area 10.2m
2Test-results is as shown in table 2.The result shows, the biocontrol effect of engineering strain viride (Trichoderma viride) R65 CCTCC N0:M 208265 spore liquid preparations has improved 23% than original strain viride (T.viride) W512, and tomato yield improves 29.8% after using the engineering strain preparation.
Table 2 tomato booth single factor experiment integrated data
| Handle | Plant height cm | The thick cm of stem | Biocontrol effect % | Cell production kg | Comparison photograph ± % |
| W512 | 217.4bcAB | 1.239a | 72％b | 202.3b | 4.0 |
| R65 | 242.6aA | 1.350a | 95％a | 252.4a | 29.8 |
| Contrast | 210.2bcB | 1.230a | - | 194.5b | - |
The plastic tent cucumber single factor experiment: test site is identical with the tomato test.For investigate the biocontrol effect of two strains tested preparations comprehensively, analyze the growth-promoting functions of engineering strain viride (Trichoderma viride) R65 CCTCC N0:M 208265 preparations under the state of land for growing field crops, this canopy is live in by the end of August, for the examination cucumber variety is " prosperous educating No. 35 ", has Powdery Mildew, brown spot slightly to take place during test in the canopy.The spray medicine cycle is identical with the tomato single factor experiment with method.Sub-district area 10.5m
2Test-results such as table 3.The result shows that the biocontrol effect of engineering strain preparation has improved 18% than original strain, and cucumber yield improves 21.9% after using engineering strain engineering strain preparation.
Table 3 cucumber booth single factor experiment integrated data
| Handle | Plant height cm | The thick cm of stem | Biocontrol effect % | Cell production kg | Comparison photograph ± % |
| W512 | 294.4ab | 0.910a | 74％b | 263.2b | 4.2 |
| R65 | 317.0a | 1.073a | 92％a | 307.8a | 21.9 |
| Contrast | 281.6b | 1.004a | - | 252.6b | - |
Claims (6)
1. a trichoderma viride engineering bacterium, contained expression inscribe chitin gene, this bacterium called after viride (Trichoderma viride) R65, and be preserved in 2008 months 29 days on the 12nd that " Chinese typical culture collection " center ", preserving number is CCTCC NO:M 208265.
2. viride engineering bacteria as claimed in claim 1, it is characterized in that: the morphological specificity of described viride engineering bacteria is: on the PDA substratum, its bacterium colony is the velvet-like of colourless or light color, and later stage bacterium colony surface begins to occur green, the visible concentricity in gonimoblast bundle district; Mycelia is separated, and branch is complicated; To giving birth to or the alternate branch, can continue branch again on the branch on the mycelia brachyplast, branch is acute angle or intimate right angle, Shu Sheng, to giving birth to or the Dan Shengmo branch; Conidium is born in succession by final terminal branch, by mucus their conglomerates or subglobose spore submanifold; Conidium is subsphaeroidal, ellipse or round shape, transparent or bright green.
3. the application of the described viride engineering bacteria of claim 1 in controlling vegetables.
4. application as claimed in claim 3 is characterized in that: described viride engineering bacteria is implemented with the biological prevention and control agent form; Wherein said biological prevention and control agent prepares with following method: viride (Trichoderma viride) R65 CCTCC NO:M 208265 is inoculated in the PDA solid medium, cultivated 4～5 days for 28～30 ℃, with sterilized water or cold water spore is washed, collecting spore and regulating its concentration is 1～5 * 10
7Individual/ml, obtain the spore suspension preparation.
5. as claim 3 or 4 described application, it is characterized in that: described viride engineering bacteria enforcement to the method for controlling vegetables is: under 20～30 ℃ of envrionment temperatures, be 1～5 * 10 with concentration
7The spore liquid of individual/ml is irritated root to the potted plant vegetable seedling that was unearthed 10 ± 1 days, handles 1～2 time, every 10 days once; Or be 1～5 * 10 with concentration
7The spore liquid of individual/ml sprays the vegetables of booth field planting, handles 3～6 times, every 10 days once.
6. application as claimed in claim 3 is characterized in that: described vegetables are cucumber or tomato.
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Cited By (5)
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| CN101942401B (en) * | 2009-12-31 | 2012-08-22 | 江南大学 | Strain for producing chitinase and method for producing chitinase in high yield |
| CN105154336A (en) * | 2015-09-12 | 2015-12-16 | 哈密信合棉业股份有限公司 | Trichoderma viride XJ-3 strain and method for preparing cotton stalk rotten organic fertilizer by using same |
| CN105145639A (en) * | 2015-10-13 | 2015-12-16 | 苏州科大微龙农业科技有限公司 | Cellulase organic copper mixture for controlling root rot of tomato phytophthora |
| CN108441458A (en) * | 2017-11-22 | 2018-08-24 | 东北农业大学 | Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application |
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| RU2750957C2 (en) * | 2020-09-24 | 2021-07-07 | Александр Васильевич Кураков | Trichoderma viride strain - producer of soyasaponin with antifungal and growth-stimulating activity against plants and earthworms |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131305C (en) * | 2001-02-13 | 2003-12-17 | 中国科学院遗传研究所 | Trichoderma chitinase and gene for expressing it |
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- 2009-01-15 CN CN2009100138434A patent/CN101492646B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101942401B (en) * | 2009-12-31 | 2012-08-22 | 江南大学 | Strain for producing chitinase and method for producing chitinase in high yield |
| CN105154336A (en) * | 2015-09-12 | 2015-12-16 | 哈密信合棉业股份有限公司 | Trichoderma viride XJ-3 strain and method for preparing cotton stalk rotten organic fertilizer by using same |
| CN105154336B (en) * | 2015-09-12 | 2018-03-23 | 哈密信合棉业股份有限公司 | A kind of Trichoderma viride XJ 3 and its method for preparing cotton stalk decomposed manure |
| CN105145639A (en) * | 2015-10-13 | 2015-12-16 | 苏州科大微龙农业科技有限公司 | Cellulase organic copper mixture for controlling root rot of tomato phytophthora |
| CN108441458A (en) * | 2017-11-22 | 2018-08-24 | 东北农业大学 | Effectively genetic engineering bacillus amyloliquefaciens of prevention gray mold and preparation method thereof and its application |
| CN108441458B (en) * | 2017-11-22 | 2021-05-25 | 东北农业大学 | Genetically engineered bacillus amyloliquefaciens capable of effectively preventing and treating gray mold as well as preparation method and application thereof |
| CN112695054A (en) * | 2021-01-21 | 2021-04-23 | 南京师范大学 | Construction method and application of high-expression chitinase endophytic fungus Liquidambaris phomopsis |
| CN112695054B (en) * | 2021-01-21 | 2023-02-10 | 南京师范大学 | Construction method and application of high-expression chitinase endophytic fungus Liquidambaris phomopsis |
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| CN101492646B (en) | 2010-09-15 |
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