CN102212485B - Trichoderma longibrachiatum and application thereof in preventing and treating vegetable diseases - Google Patents
Trichoderma longibrachiatum and application thereof in preventing and treating vegetable diseases Download PDFInfo
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Abstract
The invention discloses trichoderma longibrachiatum and application thereof to preventing and treating vegetable diseases. The collection number of the trichoderma longibrachiatum is CGMCC No.4708. The trichoderma longibrachiatum can be applied to preventing and treating vegetable diseases. The trichoderma longibrachiatum Gu 4-5 (or a preparation using the trichoderma longibrachiatum Gu 4-5 as anactive ingredient) is environment-friendly, can inhibit medicament resistance development of disease pathogeny, contributes to popularization of green food and organic agriculture, has low cost and no pollution, and is safe for vegetable crops. Antagonistic microorganisms are introduced into soil, so fungal diseases can be effectively prevented and treated through biological prevention and control, the environment can be effectively improved, an agroecological system can be maintained to be balanced, and strong support is provided for sustainable development.
Description
Technical field
The present invention relates to the mould and application in the control vegetable disease of a kind of long handle wood.
Background technology
At present, China's growing vegetables area surpasses 15,000 ten thousand mu, and wherein booth vegetable and facilities horticulture plant are above 2,010 ten thousand mu.Because the expansion and the continuous cropping continuous cropping of vegetable greenhouse cultivated area, for providing of disease and pest favourable ecological condition.The vegetables blight, gray mold, multiple fungal disease such as damping-off generally takes place in growing vegetables, takes place to cause the underproduction significantly when serious, is the peasant in the thorny problem of growing seedlings, often running in the planting process.
Blight be one type of seedling phase from root infect, the entozoic whole-plant disease of vascular bundle, but generally less apparent disease of seedling phase.The cause of disease of blight is one type of fungi (Fusarium oxysporum Schlecht) that is called sharp sickle spore.The diseased plant basal part of stem surface mould layer of finding pink is mycelium, conidium and the chlamydospore of germ.Its conidium is divided big or small amphitypy again: macroconidium is sickleshaped or crescent seemingly, light color, base portion pediculated cells, 3～5 diaphragms, most 3 separations, 30～60 * 3.5～5 microns of sizes; The microconidium oblong, light color, unit cell or a tabula is arranged, 5～26 * 2～4.5 microns of sizes.Point sickle spore bacterium is a common fungi in one type of soil, has plenty of saprophyticly, but much is the vascular bundle bacterial parasite of plant, causes blight.Wilt is mainly survived the winter in invalid body and soil with mycelium and chlamydospore, and germ has indomitable vitality in soil, can survive 6～7 years.Under the suitable condition of humiture, mycelium can produce a large amount of conidiums, propagates through irrigation water, soil cultivation, subterranean pest-insect or soil nematodes.Invade from root wound or natural breach or root cap behind the spore-germination.After the intrusion, expand during beginning slowly, after getting into fascicular conduit, propagation rate is accelerated.Germ can secrete some lytic enzymes in conduit, cause the many gelatinoids of accumulation in the conduit and stop up conduit, and germ can also excrete poison and poison the host simultaneously, thereby makes that the infected plant performance is wilted, withered symptom.The characteristics of blight are (promptly so-called incubation period) chronic from infecting to falling ill, and generally only once infect in season a growth, rarely infect generation again.The generation of blight and following condition are closely related: it is heavier that (1) belongs to melon continuous cropping morbidity of the same race together, like cucumber, muskmelon continuous cropping, and wax gourd, the continuous cropping of joint melon; (2) acid soil (like potential of hydrogen 5.5～6.5), soil too ventilate, use the organic manure that do not become thoroughly decomposed etc. all helps morbidity; (3) higher soil temperature and temperature, the plant transpiration amount is big, accelerates PD (general soil temperature is just beginning morbidity more than 15 ℃, temperature is suitable for morbidity most for 24～28 ℃, and the high temperature more than 30 ℃ has certain restraining effect to germ); (4) the melon patch morbidity that subterranean pest-insect is many is also heavier.
Gray mold is the very big disease of a kind of hazardness in the northern China vegetable-growing area.The popular of gray mold often takes place in vegetables such as the tomato of protection facility cultivations such as plastic greenhouse, greenhouse, little shed, capsicum, cucumber, Kidney bean, and the underproduction reaches more than 20～30% when serious.The cause of disease of graw mold of tomato is Botrytis cinerea bacterium (Botrytis cinerea Pers.), belongs to a kind of fungi of imperfect fungi.Conidiophore is elongated, has to separate and branch, and ash is to beige, and Cheng Congcong host's epidermis grows, and size is 280～550 * 12～24 microns.Conidium is subsphaeroidal or avette, and unicellular, light color, size are 9～15 * 6.5～10 microns.Germ also can produce chocolate, difform sclerotium.The germ host range is wider, except that infecting solanaceous vegetable, also can infect crops such as melon, wild cabbage, Kidney bean, lettuce, onion, apple.Germ is mainly examined with invalid body or is retained in the soil with mycelium or germ and survives the winter, and becomes the bacterium source of infecting at the beginning of next growth season.Also can survival all the year round in the protection cultivating facility the southern germ of China.The conidium of germ can be propagated through wind and rain, insect even farming operation, sprouts when condition is suitable, how invades from wound or aging, necrotic tissue.Grow a large amount of new conidiums again after just infecting morbidity, can constantly infect again through propagating.
Produce at present and go up the control of many employing chemical agents, but use agricultural chemicals to cause pathogenic bacteria to produce resistance for a long time in large quantities, and the havoc agroecosystem, pollution caused to environment.
Summary of the invention
The purpose of this invention is to provide the mould and application in the control vegetable disease of a kind of long handle wood.
Long handle wood provided by the invention mould (Trichoderma longibrachiatum); It is mould also to can be described as long shoot wood, and name is called Gu 4-5, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 28th, 2011 and (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4708.Long handle wood mould (Trichoderma longibrachiatum) Gu 4-5CGMCC No.4708 is called for short the mould Gu 4-5 of long handle wood.
The mould Gu 4-5 of long handle wood can be used for preventing and treating vegetable disease.
Said vegetable disease can be at least a disease that causes in the following pathogenic bacterium: melon scab cladosporium sp (Cladosporium cucumerinum), Fusarium oxysporum (Fusarium oxysporum), melon rod spore mould (Corynespora cassiicola), Phytophthora capsici (Phytophthora capsici), Botrytis cinerea bacterium (Botrytis Cinerea), dry thread Pyrenomycetes (Rhizoctonia solanii) and sclerotium sclerotinite (Sclerotinia sclerotiorumm).
The present invention also protects a kind of preparation of controlling plant diseases, and its activeconstituents is the mould Gu 4-5 of long handle wood.
It also can comprise carrier said preparation, preferred pesticide field commonly used and be the inert carrier biologically.Said carrier can be solid carrier or liquid vehicle.Said solid carrier is mineral material, vegetable material or macromolecular compound; Said mineral material can be at least a in clay, talcum, kaolin, smectite, white carbon, zeolite, silica and the zeyssatite.Said vegetable material can be at least a in Semen Maydis powder, bean powder and the starch.Said macromolecular compound can be Z 150PH and/or polyglycol.Said liquid vehicle can be organic solvent, vegetables oil, MO or water.Said organic solvent can be decane and/or dodecyl.In the said preparation, said long handle wood is mould can be existed with the form of the mixture of the filtrating of the viable cell cultivated, cell culture or cell and filtrating.Said viable cell can be conidium, chlamydospore, mycelia or contains conidium and the mycelial form of mycelia, is preferably conidium or chlamydosporic form.Said preparation can adopt multiple formulation, like liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules, is preferably and is suitable for spraying the formulation of using.As required, also can add tensio-active agent (like polysorbas20, tween 80 etc.), tackiness agent, stablizer (like inhibitor), pH regulator agent etc. in the said preparation.
The conidium quantity of bacterial strain Gu 4-5 in the preparation of the present invention changes with formulation and the method for using.In the solid preparation, conidial quantity is (1 * 10
5)-(1 * 10
8) individual spore/gram, preferred (1 * 10
6)-(1 * 10
7) individual spore/gram.For liquid preparation, conidial content can be (2.0 * 10 in the flushing liquor after the dilution
4)-(5.0 * 10
7) individual spore/mL, preferred 10
7Individual spore/mL.
Said preparation can be used with other chemical pesticide that is fit to, thereby reduces the consumption of chemical pesticide agent, reduces the pollution to environment.
Said preparation can be used for preventing and treating vegetable disease.
Said vegetable disease can be at least a disease that causes in the following pathogenic bacterium: melon scab cladosporium sp (Cladosporium cucumerinum), Fusarium oxysporum (Fusarium oxysporum), melon rod spore mould (Corynespora cassiicola), Phytophthora capsici (Phytophthora capsici), Botrytis cinerea bacterium (Botrytis Cinerea), dry thread Pyrenomycetes (Rhizoctonia solanii) and sclerotium sclerotinite (Sclerotinia sclerotiorumm).
The mould Gu 4-5 of long handle of the present invention wood (or be formulations of active ingredients with the mould Gu 4-5 of long handle wood) is environmentally friendly; The resistance and the development of drug resistance that can suppress the disease cause of disease; Help the popularization of green food and Organic farming, and cost is low, pollution-free, to vegetable crop safety.In soil, introduce antagonistic microbe, can prevent and treat fungal disease effectively and can effectively improve environment, keep the agroecosystem balance, powerful backing is provided for walking sustainable development path through biological control.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used black spot pathogenic bacterium are melon scab cladosporium sp (Cladosporium cucumerinum) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 36060.Used blight pathogenic bacterium are Fusarium oxysporum (Fusarium oxysporum) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 36478.Used brown spot pathogenic bacterium are melon rod spore mould (Corynespora cassiicola) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC36068.Used eqpidemic disease pathogenic bacterium are Phytophthora capsici (Phytophthora capsici) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 37401.Used gray mold pathogenic bacterium are Botrytis cinerea bacterium (Botrytis Cinerea) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 36041.Used hypochnus pathogenic bacterium are dry thread Pyrenomycetes (Rhizoctonia solanii) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 36457.Used sclerotium disease pathogenic bacterium are sclerotium sclerotinite (Sclerotinia scleroiorumm) among the embodiment, available from Chinese agriculture microbial strains preservation administrative center, deposit number ACCC 30096.
Acquisition and the preservation of embodiment 1, long handle wood mould (Trichoderma longibrachiatum) Gu 4-5
One, sampling
From Beijing, Hebei, Xinjiang, Shandong, Hubei etc. gather soil sample.
Two, strains separation
Adopt the coating method isolated strains.Each soil sample takes by weighing 10g, pours into respectively to be added with in the aseptic 500ml triangular flask of a little tween 80, granulated glass sphere and 100ml zero(ppm) water, shakes up, and label shook 30 minutes with shaking table.Inhale 5ml soil suspension liquid in joining the 250ml triangular flask that contains the 45ml sterile purified water with microsyringe then, shake up, from the 250ml triangular flask, inhale 1ml again and join in the test tube that contains the 9ml sterile purified water, carry out doubling dilution, sample gradient dilution to 10
-4With 10
-5Each sample inhale 0.1ml and drip in PDA dull and stereotypedly, coating was cultivated 48-72 hour for 28 ℃, selected and produced the sporogenic bacterium colony of green sub, carried out the inclined-plane inoculation, treat that bacterium colony grows after, will transfer on the PDA flat board, separate, purifying, up to obtaining pure culture.Place 4 ℃ of preservations of refrigerator.
Three, bacterial strain screening
1, plate antagonism screening
At the dull and stereotyped center inoculation of PDA Trichoderma piece, on the circumference about radius 2.5cm, 3 equivalent various pathogenic bacteria of equidistant inoculation as one group, have three groups with per three kinds of different pathogenic bacterias, establish three repetitions for every group.With marking pen marked strain number, in order to observed and recorded.The petridish that inoculation is good is put in 28 ℃ the incubator and cultivates about 7-10d.The next day, take pictures to the experiment of being done, and measures pathogenic bacteria and point to wood mould radius R and r.Morphological specificity between the mould and pathogenic bacteria of record wood.After germ and the wooden mould handing-over, observe encirclement, inhibition that pathogenic bacteria falls to Trichoderma, invade and capture its nutrition spatial process.The screening trichoderma strain, carry out according to following principle: (1) speed of growth is wooden mould apparently higher than pathogenic bacteria, can fight for nutrition with pathogenic bacteria, thereby significantly suppress growth of pathogenic bacteria; (2) and space, the 0.2cm left and right sides is arranged between the pathogenic bacteria, promptly produced antibacterial band, mould certain antibiotics that in metabolic process, produced of wood has been described.The result sees table 1.
Plate face-off record between table 1 trichoderma strain and the 9 kinds of pathogenic bacterias
| Strain number | The cotton Huang withers | The eggplant Huang withers | The cereal reaping hook | Tomato gray mould | Cucumber is withered | Watermelon is withered | The cucumber anthrax | Genseng is upright withered | Cotton standing dead |
| Gu?45 | +++ | +++ | ++ | +++ | ++ | +++ | +++ | ++ | ++ |
Annotate: trichoderma strain is to the growth of pathogenic bacteria restraining effect, by by force to a little less than, be divided into +++, ++ ,+,-four grades.
2, potted plant live body screening
(1) preparation of Trichoderma suspension
On the wooden mould plate that is re-seeded into the PDA substratum that the plate antagonism is filtered out, cultivate about 5d and grow spore.With spore under the writing brush brush, being made into concentration is 10
7The bacteria suspension of/ml.
(2) wooden mould preparation of fermentation liquid
In the wooden mould plate that is inoculated in the PDA substratum respectively that filters out, cultivate after 5 days, have in being transferred in the triangular flask of 250ml of 100ml nutrient solution, put into shaking table and cultivated 3 days for 30 ℃.
(3) screening
To supply examination Trichoderma suspension and contrast medicament to prepare by experimental concentration respectively; The vaccination ways of black spot pathogenic bacterium, brown spot pathogenic bacterium adopts the spore suspension spray inoculation; The vaccination ways of gray mold pathogenic bacterium, hypochnus pathogenic bacterium, sclerotium disease pathogenic bacterium adopts lawn blade face inoculation method, blight pathogenic bacterium to adopt the radicle inoculation method, and the eqpidemic disease pathogenic bacterium are adopted the leaf lawn blade face inoculation method that exsomatizes.
At first execute the Trichoderma suspension; Gray mold pathogenic bacterium, hypochnus pathogenic bacterium, black spot pathogenic bacterium, brown spot pathogenic bacterium, sclerotium disease pathogenic bacterium, eqpidemic disease pathogenic bacterium are adopted the mode that sprays; Inoculate pathogenic bacteria after waiting to spray back 2h; The blight pathogenic bacterium are soaked the mode of dispenser, the cultivation of preserving moisture after the inoculation.
Carry out flat board face-off test with separating trichoderma strain and 9 kinds of pathogenic fungies that generally take place of obtaining; Filter out above-mentioned to there being the wooden mould of obvious antagonistic action that 7 kinds of pathogenic bacterias are carried out indoor pot experiment; Filter out a strain long handle trichoderma strain 3 kinds of diseases such as eqpidemic disease pathogenic bacterium, blight pathogenic bacterium, gray mold pathogenic bacterium are all had the better prevention effect; This bacterial strain has how anti-characteristic, is fit to promote produce.
The trichoderma strain that filters out is numbered Gu 4-5 bacterial strain.
Four, identification of strains
(1) morphology of bacterial strain is identified
With inoculation on the PDA substratum, 26 ℃ of constant temperature culture 3 days, record colonial morphology.The bacterium cake of cut-off footpath 2em is placed in the petridish that is covered with two-layer moistening filter paper, manufactures slide after 4 days, and observed and recorded spore shape and conidial fructification are measured spore (30) and sporophore width etc.
The morphological specificity of Gu 4-5 bacterial strain: colony diameter is dark culturing on PDA, and 25-30 ℃ of cultivation is 65-70mm, cultivates 13-70mm at 40 ℃, and under the situation of 30-40 ℃ of cultivation, the 24h endoconidium occurs, and 30-35 ℃ of a large amount of the appearance, does not occur at 20 ℃.35 ℃ of dark culturing 66h on PDA, conidium is covered with planar surface, forms concentric(al) circles in 30 ℃ of conidiums; The a large amount of blackish greens of conidium, variegated sometimes, the spot of adularescent; And often have unconspicuous yellow mycelia to be distributed in a large amount of conidial surfaces, and produce xanchromatic diffustivity pigment, mainly produce down at 30 ℃; Also can produce at 20 ℃ and 35 ℃, not produce under 40 ℃ of situation.Bacterium colony 20 ℃ of alternation of light and darkness on CMD cultivated for 1 week, and conidium generates along aerial hyphae, or successive merges into the aggregate of cushion.Conidiophore on CMD by thick main shaft from stretching out a little until the top, single bottle stalk, secondary branch tower shape distribution is often to life.Bottle is upright then gives birth on secondary branch, not verticillate.The main shaft width of conidiophore is top 1.2-2.2um, base portion 3.2-4.7 μ m.Bottle stalk Dan Sheng, frequent colyliform, cylindricality, verticillate or squat and give birth to the bottle stalk, expand at the middle part, straight or be bent to irregular.Between give birth to the bottle stalk and see more.Conidium is green on CMD, and is microscler to long oval, smooth.
(2) Molecular Identification of bacterial strain
The zellglas on the dull and stereotyped upper berth of PDA is inoculated into strains tested (Gu 4-5) on the zellglas then, and bacterial classification inoculation is in plate central authorities, 28 ℃ of dark culturing 72h.Adopt the CTAB method to carry out DNA extraction, adopt the DNAOUT of pool, sky, high and new technology industrial development zone, Mianyang genetically engineered manufactured to extract test kit, carry out the ITS amplification according to (1990) conditions such as White,, the PCR product send the order-checking of handsome Bioisystech Co., Ltd.The ITS information (seeing the sequence 1 of sequence table) of order-checking bacterial strain is carried out BLAST analyze, provide the qualification result of bacterial strain.BLAST is identified not high bacterial strain information, ISTH website (the INTERNATIONAL SUBCOMMISSION ON TRICHODERMA AND HYPOCREA TAXONOMY of coincidence rate; Www.isth.info) and NCBI (National Center for Biotechonology Information; Www.ncbi.nlm.nih.gov) select the ITS sequence for use; Carry out cluster analysis with ClustalX (1.81) and Molecular Evolutionary Genetics Analysis Version 3.1 softwares; Combining form analytical results and sequence cluster analysis result identify kind with trichoderma strain.
Through sequence alignment, sequence comparative result and morphology qualification result, bacterial strain Gu 4-5 is accredited as long handle wood mould (Trichoderma longibrachiatum).The mould Gu 4-5 of long handle wood is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 28th, 2011, and preserving number is CGMCC No.4708.The mould Gu 4-5 of long handle wood is subordinate to Trichoderma (Trichoderma), sticking spore mushroom (Gliolosporae), hyphomycetales (Hyphomycetales), hyphomycetes (Hyphomycetes), Deuteromycotina (Deuteromycotina).
Embodiment 2, the application of the mould Gu 4-5 of long handle wood in the control vegetable disease
Through the artificial inoculation method, measure the inhibition situation of the mould Gu 4-5 of long handle wood respectively to black spot pathogenic bacterium, blight pathogenic bacterium, brown spot pathogenic bacterium, eqpidemic disease pathogenic bacterium, gray mold pathogenic bacterium, hypochnus pathogenic bacterium and sclerotium disease pathogenic bacterium disease.
3% jinggangmeisu aqua: Zhejiang Tonglu Huifeng Bio-Chemical Co., Ltd..50% HSDB 6915: auspicious chemical industry ltd on the Hebei.75% m-tetrachlorophthalodinitrile: Xinyi Feng Le chemical industry ltd.40% lucky star EC500: Du Pont China Holding Co., Ltd..40% phonetic mould amine suspension agent: Yantai Keda Chemical Co., Ltd..70% thiophanate methyl: Huayang Agricultural Chemial Group Co Ltd, Shandong.Jinggangmeisu solution: 0.01g 3% jinggangmeisu aqua is dissolved in 20ml water, obtains jinggangmeisu solution.HSDB 6915 solution: 0.01g 50% HSDB 6915 is dissolved in 20ml water, obtains HSDB 6915 solution.
M-tetrachlorophthalodinitrile solution: 0.01g 75% m-tetrachlorophthalodinitrile is dissolved in 20ml water, obtains m-tetrachlorophthalodinitrile solution.Lucky star EC solution: 0.01g 40% lucky star EC500 is dissolved in 20ml water, obtains lucky star EC solution.Phonetic mould amine suspension solution: the phonetic mould amine suspension agent of 0.01g40% is dissolved in 20ml water, obtains phonetic mould amine suspension solution.Thiophanate methyl solution: 0.01g 70% thiophanate methyl is dissolved in 20ml water, obtains thiophanate methyl solution.
One, the preparation of the mould Gu 4-5 bacteria suspension of long handle wood
Mould Gu 4-5 is inoculated on the PDA culture medium flat plate with long handle wood, cultivates about 5d and grows spore, and with spore under the writing brush brush, using sterilized water to be made into concentration is 10
7The bacteria suspension of individual/ml.
Two, the mould Gu 4-5 of long handle wood tests epidemic prevention and control
Seedling phase pepper plant is divided into three groups, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at leaf on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at leaf on atomizer 50% HSDB 6915 solution;
The 3rd group (negative control): evenly be sprayed at leaf on atomizer sterilized water;
Blade after above-mentioned two or three group handled is preserved moisture and was placed 1 hour; Then eqpidemic disease pathogenic bacterium flat board is beaten diameter 0.5cm bacterium sheet and be seeded on the leaf, 2 leaves of every strain capsicum inoculation, bacterium sheet of every leaf inoculation.Treat negative control when morbidity (behind the inoculation pathogenic bacterium 48 hours, promptly pathogenic bacteria grows surely, causes an inoculation point blade to be compared with healthy position, pathologic occurs and changes), the lesion area situation of three groups of pepper plant blades of investigation records on blade.
Three, the mould Gu 4-5 of long handle wood tests sheath blight disease controlling
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cotyledon, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at cotyledon on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at cotyledon on atomizer jingganmycin solution;
The 3rd group (negative control): evenly be sprayed at cotyledon on atomizer sterilized water;
Blade after above-mentioned three groups handled is preserved moisture and was placed 6 hours; Then hypochnus pathogenic bacterium flat board is beaten diameter 0.5cm bacterium sheet and be seeded on the leaf, 2 leaves of every strain inoculation, bacterium sheet of every cotyledon inoculation; Preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(inoculation pathogenic bacterium 72 hours, promptly pathogenic bacteria grows on blade surely, causes inoculation point blade to be compared with healthy position, pathologic occurs and changes) are investigated three groups of cucumber plant leaf spot lesion area situation when treating the negative control morbidity.
Four, the mould Gu 4-5 of long handle wood is to gray mold control experiment
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cotyledon, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at cotyledon on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at cotyledon on atomizer m-tetrachlorophthalodinitrile solution;
The 3rd group (negative control): sterilized water evenly is sprayed on the cotyledon with atomizer, and every leaf sprays;
Blade after above-mentioned three groups handled is preserved moisture and was placed 6 hours; Then the dull and stereotyped bacterium sheet of beating diameter 0.5cm of gray mold pathogenic bacterium is seeded on the leaf 2 leaves of every strain inoculation, bacterium sheet of every cotyledon inoculation; Preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(treat that pathogenic bacteria grows surely on blade, cause inoculation point blade to be compared, pathologic occurs and change) when treating the negative control morbidity, investigate three groups of cucumber plant leaf spot lesion areas with healthy position.
Five, the mould Gu 4-5 of long handle wood is to brown spot control experiment
After cultivating six days on the PDA flat board, with writing brush with the spore brush down, being made into concentration is 10 with the brown spot pathogenic bacterium
6The spore suspension of individual/ml, i.e. brown spot pathogenic bacterium bacteria suspension.
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cucumber cotyledons, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at cotyledon on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at cotyledon on atomizer m-tetrachlorophthalodinitrile solution;
The 3rd group (negative control): evenly be sprayed at cotyledon on atomizer sterilized water;
Blade after above-mentioned three groups handled is preserved moisture and was placed 6 hours; Then brown spot pathogenic bacterium bacteria suspension is sprayed onto on the cotyledon of handling uniformly, preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(treat that pathogenic bacteria grows surely on blade, cause inoculation point blade to be compared, pathologic occurs and change) when treating the negative control morbidity, investigate three groups of cucumber plant leaf spot lesion areas with healthy position.
Six, the mould Gu 4-5 of long handle wood is to the control experiment of black spot
After cultivating six days on the PDA flat board, with writing brush with the spore brush down, being made into concentration is 10 with the black spot pathogenic bacterium
6The spore suspension of individual/ml, i.e. black spot pathogenic bacterium bacteria suspension.
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cucumber cotyledons, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at cotyledon on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at cotyledon on atomizer lucky star EC solution;
The 3rd group (negative control): evenly be sprayed at cotyledon on atomizer sterilized water;
Blade after above-mentioned three groups handled is preserved moisture and was placed 6 hours; Then black spot pathogenic bacterium bacteria suspension is sprayed onto on the cotyledon of handling uniformly; Preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(treat that pathogenic bacteria grows surely on blade, cause inoculation point blade to be compared, pathologic occurs and change) when treating the negative control morbidity, investigate three groups of cucumber plant leaf spot lesion areas with healthy position.
Seven, the mould Gu 4-5 of long handle wood suppresses experiment to sclerotium pathogenic bacteria live body
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cotyledon, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: evenly be sprayed at cotyledon on atomizer the mould Gu 4-5 bacteria suspension of long handle wood;
Second group (positive control): evenly be sprayed at cotyledon on atomizer phonetic mould amine suspension solution;
The 3rd group (negative control): sterilized water evenly is sprayed on the cotyledon with atomizer, and every leaf sprays;
Blade after above-mentioned three groups handled is preserved moisture and was placed 6 hours; Then the dull and stereotyped bacterium sheet of beating diameter 0.5cm of sclerotium disease pathogenic bacterium is seeded on the leaf 2 leaves of every strain inoculation, bacterium sheet of every cotyledon inoculation; Preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(treat that pathogenic bacteria grows surely on blade, cause inoculation point blade to be compared, pathologic occurs and change) when treating the negative control morbidity, investigate three groups of cucumber plant leaf spot lesion areas with healthy position.
Eight, the mould Gu 4-5 of long handle wood is to blight control experiment
After cultivating six days on the PDA flat board, with writing brush with the spore brush down, being made into concentration is 10 with the cucumber fusarium axysporum pathogenic bacterium
6The spore suspension of individual/ml, i.e. blight pathogenic bacterium bacteria suspension.
The cucumber seeds kind in the vesicle dish, is divided into three groups to the open and flat phase of cotyledon, handles (every group is provided with 5 parallel processing) respectively as follows:
First group: with the mould Gu 4-5 bacteria suspension radicle immersion treatment of long handle wood;
Second group (positive control): adopt thiophanate methyl solution radicle immersion treatment;
The 3rd group (negative control): sterilized water radicle immersion treatment;
Handle the back radicles with above-mentioned three groups and preserve moisture and placed 6 hours, then cucumber fusarium axysporum pathogenic bacterium bacteria suspension is soaked Baconic, preserved moisture in 24 hours behind the inoculation pathogenic bacterium.(treat the variable color of cucumber plant vascular bundle, the wilting symptom occurs, investigate three groups of cucumber fusarium axysporum sickness rate situation when treating the negative control morbidity.
The result of step 2 to eight sees table 2.
The mould Gu 4-5 of table 2 long handle wood is to the main disease-controlling effect of vegetables
Claims (9)
1. long handle wood mould (Trichoderma longibrachiatum), its deposit number is CGMCC No.4708.
2. the mould application in the control vegetable disease of the said long handle wood of claim 1; Said vegetable disease is at least a disease that causes in the following pathogenic bacterium: melon scab cladosporium sp (Cladosporium cucumerinum), Fusarium oxysporum (Fusarium oxysporum), melon rod spore mould (Corynespora cassiicola), Phytophthora capsici (Phytophthora capsici), Botrytis cinerea bacterium (Botrytis Cinerea), dry thread Pyrenomycetes (Rhizoctonia solanii) and sclerotium sclerotinite (Sclerotinia sclerotiorumm).
3. the preparation of a controlling plant diseases, its activeconstituents is that the said long handle wood of claim 1 is mould.
4. preparation as claimed in claim 3 is characterized in that: it also comprises carrier; Said carrier is solid carrier or liquid vehicle; Said solid carrier is mineral material, vegetable material or macromolecular compound; Said mineral material is at least a in clay, talcum, kaolin, smectite, white carbon, zeolite, silica and the zeyssatite; Said vegetable material is at least a in Semen Maydis powder, bean powder and the starch; Said macromolecular compound is Z 150PH and/or polyglycol; Said liquid vehicle is organic solvent or water; Said organic solvent is vegetables oil, MO, decane and/or dodecyl.
5. like claim 3 or 4 described preparations, it is characterized in that: in the said preparation, the form of the mixture of mould filtrating or cell and the filtrating with the viable cell cultivated, cell culture of said long handle wood exists.
6. preparation as claimed in claim 5 is characterized in that: said viable cell is conidium, chlamydospore, mycelia or contains conidium and the mycelial form of mycelia.
7. preparation as claimed in claim 6 is characterized in that: said viable cell is conidium or chlamydosporic form.
8. like claim 3 or 4 described preparations, it is characterized in that: the formulation of said preparation is liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
9. the application of arbitrary described preparation in the control vegetable disease in the claim 3 to 8; Said vegetable disease is at least a disease that causes in the following pathogenic bacterium: melon scab cladosporium sp (Cladosporium cucumerinum), Fusarium oxysporum (Fusarium oxysporum), melon rod spore mould (Corynespora cassiicola), Phytophthora capsici (Phytophthora capsici), Botrytis cinerea bacterium (Botrytis Cinerea), dry thread Pyrenomycetes (Rhizoctonia solanii) and sclerotium sclerotinite (Sclerotinia sclerotiorumm).
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| EP2910127A1 (en) * | 2014-02-21 | 2015-08-26 | Koppert B.V. | Bioactive protein, use thereof and method for its production |
| CN108342330A (en) * | 2018-05-09 | 2018-07-31 | 中国科学院天津工业生物技术研究所 | One plant of long shoot trichoderma and its application with broad spectrum antibacterial performance |
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