CN105132296B - A kind of hook-shaped trichoderma strain and its application - Google Patents
A kind of hook-shaped trichoderma strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of hook-shaped trichoderma strain and its application, the bacterial strain is hook-shaped trichoderma strain Tham 21, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.10923.The application of biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent prepared by hook-shaped trichoderma strain Tham 21 in root-knot nematode is prevented.Hook-shaped 21 bacterial strains of Trichoderma Tham are the fungi being separated to from soil, harmless to crop safety, and have preventive and therapeutic effect to root-knot nematode, in the application with security, suitable for the biological control of root-knot nematode.Hook-shaped 21 bacterial strain fermentation liquors of Trichoderma Tham reach more than 98% to the lethality of root-knot nematode second instar larvae, and prevention effect in the greenhouse reaches 87.2%, and prevention effect is high and stablizes, suitable for agricultural production demand.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of hook-shaped trichoderma strain and its application.
Background technology
Root-knot nematode (Meloidogyne) is a kind of omnivorousness plant pathogeny line insect of height specialized form.It is known to endanger vegetable
The nematode of dish mainly has peanut root-knot nematode, M hapla, Meloidogyne incognita, javanese root knot nematode and beet root knot
Nematode etc..Nematode host range is extensive, often endangers more than the 30 kinds of vegetable such as melon, solanaceous vegetables, beans and radish, calabash radish, lettuce, Chinese cabbage
Dish, moreover it is possible to propagate some fungies and bacterial disease.Root-knot nematode is a kind of important crop disease original, is caused every year about
Billions of losses.The root of the main infection crops of root-knot nematode, the root swelling after infecting form huge root knot, can be mutual
Connection forms beading, since root is destroyed, influences normal absorbing function, so overground part growth and development is obstructed, is invaded
Plant above ground portion performance after dye is downgraded, and leaf portion yellow is as a result small and few.Easily wilt or wither during dry weather.Connect for a long time
In the greenhouse of work, particularly even cause to have no harvest on the crop such as western muskmelon, cucumber, tomato.
Since the germ plasm resource of root-knot nematode resistant is rare, so chemical prevention is still prevention root-knot nematode in production
Important measures, such as dazomet, chloropicrin and Mobam, although these medicament desinsections are fast, can seriously reduce vegetables quality,
Environment is polluted, destroys the ecological balance, it is dangerous to people, animal during use, therefore bio-control method is increasingly subject to people's
Concern.
The content of the invention
The purpose of the present invention is to solve existing for chemical prevention in the prior art pollute environment, unsafe problem,
A kind of hook-shaped trichoderma strain and its application are provided.
To achieve the above object, the present invention provides a kind of hook-shaped trichoderma strain, the bacterial strain is hook-shaped Trichoderma bacterium
Strain Tham-21, its microbial preservation numbering is CGMCC NO.10923；Classification And Nomenclature is：Trichoderma hamatum；Protect
Hide the time：On May 27th, 2015；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism are ground
Study carefully institute；Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) is preserved in, is protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Present invention also offers biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent prepared by hook-shaped trichoderma strain.
Present invention also offers application of the biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent in root-knot nematode is prevented prepared by hook-shaped trichoderma strain.
Preferably, the root-knot nematode is Meloidogyne incognita and/or M hapla.
Present invention also offers the method that hook-shaped trichoderma strain is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, this method includes following step
Suddenly：
(1) hook-shaped trichoderma strain Tham-21 is inoculated in shaken cultivation in PDB culture mediums, obtaining spore concentration is
107-1010The spore suspension of/ml；
(2) by the spore suspension 1-2L of step (1) and diatomite 500-700g, corn flour 200-400g, wheat bran 50-
150g after mixing, carries out solid fermentation, obtains solid fermentation thing；
(3) the solid fermentation thing of step (2) is crushed, crosses 60 mesh sieves, obtain conidia powder, conidia powder and sticker, disperse
After agent, preservative mixing, biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent is prepared.
Preferably, PDB culture medium prescriptions are in step (1)：Potato 200g/L, glucose 20g/L, surplus are water.
Preferably, condition of culture is 25-28 DEG C in step (1), shaken cultivation 6-8 days.
Preferably, in step (2), solid fermentation is carried out under the conditions of 28-30 DEG C, after fermenting 6-8 days, under room temperature
It is 3-5 days dry, obtain solid fermentation thing.
Preferably, in step (3), conidia powder, sticker, dispersant, the mass ratio of preservative are 85-90：1-3：8-10：
1-2。
Preferably, in step (3), sticker is sodium carboxymethylcellulose, and dispersant is sodium lignin sulfonate, and preservative is
Streptomysin.
Beneficial effects of the present invention are：
(1) hook-shaped Trichoderma Tham-21 bacterial strains are the fungi being separated to from soil, harmless to crop safety, and to root
Tie lines worm has preventive and therapeutic effect, in the application with security, is suitable for the biological control of root-knot nematode.
(2) application method is simple and practical：Biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent is made in hook-shaped trichoderma strain, Pelleting bag is carried out to seed
Clothing, features simple and practical process.
(3) prevention effect is high and stablizes：Cause of the hook-shaped Trichoderma Tham-21 bacterial strain fermentation liquors to root-knot nematode second instar larvae
Dead rate reaches more than 98%, and prevention effect in the greenhouse reaches 87.2%, and prevention effect is high and stablizes, and is needed suitable for agricultural production
Ask.
Brief description of the drawings
Fig. 1 is the cellular morphology figure of the hook-shaped trichoderma strain Tham-21 of the present invention；
Fig. 2 is the colonial morphology figure of the hook-shaped trichoderma strain Tham-21 of the present invention.
Embodiment
The present invention is carried out with reference to embodiment and attached drawing explanation is explained in detail.
Embodiment 1
Separation, purifying and the identification of Trichoderma：
Pedotheque is uniformly taken from the tomato planting field of Shunyi, 10g soil carries out mixing system respectively with 100ml aqua sterilisas
Into suspension, gradient dilution is then carried out successively, is taken the soil of 10000 times of dilution to mix 100 μ l of liquid and is coated on PDA culture medium tablet
It is upper that (i.e. potato 200g, glucose 20g, agar 17g, distilled water are settled to 1000ml.This culture medium is sterilized at 121 DEG C,
Tablet is made in culture dish middle berth).Contain 75 μ g/ml ampicillins in PDA culture medium, inhibit bacteria growth.Culture medium is put
In 25 DEG C of cultures, after tissue block grows bacterium colony, single spore separation is carried out, picking single bacterium colony purifies in PDA culture medium, for dividing
Class is identified.According to《Fungal identification handbook》Morphology measure is carried out to bacterial strain.
Mycelia on picking colony, is put into the EP pipes for filling 300 μ l fungi lysates FPCB, when 65 DEG C of cracking 2 are small, so
Afterwards with fungal genomic DNA extracts kit (Tiangeng bio tech ltd) extraction fungal DNA, using DNA as template, with drawing
Thing ITS1, ITS4 and EF1H, EF2T carry out ITS and tef1 sequence amplifications respectively.Pcr amplification reaction system is 20 μ l, contains 10 μ
L PCR Master Mix, 1 μ l forward primers, 1 μ l reverse primers, 2 μ l DNA profilings, 0.5 μ l of Taq enzyme, 5.5 μ l of sterile water.
Amplification condition：94 DEG C of 4min, 94 DEG C of 55s, 55 DEG C of 30s, 72 DEG C of 50s, 30 circulations, 72 DEG C of 7min.Amplified production is through 1% agar
Sugared gel electrophoresis separation identification, PCR product directly carry out bidirectional sequencing.
As a result：
Referring to attached drawing 1-2, Tham-21 bacterial strains are on PDA culture medium tablet, 25 DEG C, after cultivating 3 days, colony diameter 40-
60mm, bacterium colony green.Mycelia separates, and transparent, surface is smooth, and aerial hyphae is few, a diameter of 1-7 μm.In main branch bottom raw
The secondary branch of many tubbiness.Conidiophore is in arborizations to raw or alternate, and top stigma is in doleiform, and stigma top is given birth to
Conidium group, conidiophore main shaft upper bend.For conidiophore length up to 60 μm, base portion is 5 μm wide, the production of tip lower part
Raw 1-2 verticillate ampuliform sporophores, sporophore is 6.8-13.2 μm long, and most width is 2.5-3.5 μm, and base portion is 2.0-3.0 μm.It is mitogenetic
Spore is mostly elliposoidal, and in light green, size is 2.5-4.5 × 2-4 μm.According to《Fungal identification handbook》To Tham-21 bacterial strains
Form is measured, and it is hook-shaped Trichoderma (Trichoderma hamatum) to determine the bacterial strain.
ITS and tef1 sequence amplifications are carried out respectively using primer I TS1, ITS4 and EF1H, EF2T, and PCR product is through 1% fine jade
Sepharose electrophoresis.ITS and tef1 sequences length through the amplification of sequencing analysis bacterial strain is about 570bp and 880bp.By the ITS of bacterial strain
After carrying out BLAST analyses with tef1 sequences, the results show that Tham-21 bacterial strains and hook-shaped Trichoderma (Trichoderma
Hamatum) similarity has reached 100%.It is hook-shaped Trichoderma (Trichoderma hamatum) to identify the bacterial strain.
Embodiment 2
The preparation and application of hook-shaped Trichoderma seed coat agent：
Tham-21 bacterial strains are inoculated into the triangular flask of 250ml respectively, every bottle contains PDB fluid nutrient medium 125ml,
(160rpm) is shaken in 25 DEG C to cultivate 8 days, obtains substantial amounts of mycelium and conidium.Liquid fermentation medium is potato Portugal
Grape sugar liquors (PDB) culture medium, i.e. potato 200g, glucose 20g, distilled water are settled to 1000ml.
Liquid is shaken into the pityrosporion ovale filament mixed liquor obtained by bacterium culture (fermentation), spore concentration 107A/ml), add with
Lower component：Ratio is as follows：Spore liquid 1L, diatomite 500g, corn flour 200g, wheat bran 50g, after mixing, are placed in 28 DEG C of bars
Solid secondary fermentation is carried out under part, after the 6d that ferments, solid fermentation thing is placed on natural drying 3d (fermentates under room temperature
Water content control is between 15-25%), crush, cross 60 mesh sieves, obtain conidia powder.By conidia powder and sticker (carboxymethyl cellulose
Plain sodium), dispersant (sodium lignin sulfonate), preservative (streptomysin) mixing, biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent is prepared, wherein, conidia powder,
Sticker, dispersant, the mass ratio of preservative are 85：3：10：2, then seed coat agent stirred evenly with seed, make conidia powder uniform
It is coated the weight ratio in the surface of the seed, that is, completing Pelleting Cotton seeds of the biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent to cucumber seeds, seed coat agent and seed
For 1:10(w/w).
Embodiment 3
The preparation and application of hook-shaped Trichoderma seed coat agent：
Tham-21 bacterial strains are inoculated into the triangular flask of 250ml respectively, every bottle contains PDB fluid nutrient medium 125ml,
(160rpm) is shaken in 28 DEG C to cultivate 6 days, obtains substantial amounts of mycelium and conidium.Liquid fermentation medium is potato Portugal
Grape sugar liquors (PDB) culture medium, i.e. potato 200g, glucose 20g, distilled water are settled to 1000ml.
Liquid is shaken into the pityrosporion ovale filament mixed liquor obtained by bacterium culture (fermentation), spore concentration 1010A/ml), add with
Lower component：Ratio is as follows：Spore liquid 2L, diatomite 700g, corn flour 400g, wheat bran 150g, after mixing, are placed in 30 DEG C of bars
Solid secondary fermentation is carried out under part, after the 8d that ferments, solid fermentation thing is placed on natural drying 5d (fermentates under room temperature
Water content control is between 15-25%), crush, cross 60 mesh sieves, obtain conidia powder.By conidia powder and sticker (carboxymethyl cellulose
Plain sodium), dispersant (sodium lignin sulfonate), preservative (streptomysin) mixing, biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent is prepared, wherein, conidia powder,
Sticker, dispersant, the mass ratio of preservative are 90：1：8：1, then seed coat agent stirred evenly with seed, make conidia powder uniform
It is coated the weight ratio in the surface of the seed, that is, completing Pelleting Cotton seeds of the biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent to cucumber seeds, seed coat agent and seed
For 1:10(w/w).
Embodiment 4
The nematicidal effect of hook-shaped Trichoderma Tham-21 bacterial strains：
1st, the preparation of Tham-21 bacterial strain fermentation liquors
Tham-21 bacterial strains are inoculated into the triangular flask of 250ml respectively, inoculum concentration 10%, every bottle is trained containing PDB liquid
Base 125ml is supported, (160rpm) is shaken in 25-28 DEG C and is cultivated 7 days, zymotic fluid and its 10 times, 100 times of dilutions are handled respectively
Root-knot nematode (Meloidogyne spp.), measures kill effect of the hook-shaped Trichoderma tunning to root-knot nematode.
2nd, prepare experiment and use nematode
Pick up from Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science greenhouse.Capsicum root system is taken out, is gently rinsed with water, carefully
Pieces of an egg are removed, is placed in 1% sodium hypochlorite and sterilizes 3min, then with aseptic water washing 3 times, are placed on the training containing a small amount of sterile water
Support in ware, cultivated in 25 DEG C of insulating boxs, the root-knot nematode J2 larvas once hatched are collected per 24h.
3rd, test method
On sterile Tissue Culture Plate, the zymotic fluid 1ml of various concentrations is added into hole respectively.And using sterile water as
Control, 100 μ l nematodes suspension (100 nematodes) are then added into processing and control, are placed on 24h at room temperature respectively, are observed
The death condition of root-knot nematode, calculates corrected mortality, is nematicidal effect, and each processing is repeated 3 times.Each processing does 24
A hole (sample), each experiment are repeated 3 times.
Corrected mortality (%)=(processing nemic death rate-control nemic death rate)/(1- compares nemic death rate)
× 100%
4th, result of the test：
By above-mentioned experiment, virulence effects of the different multiples fermentation liquor treatment 24h to root-knot nematode is measured, result of the test is shown in
Table 1, the results showed that different fermentations liquid multiple is different to the action effect of nematode, the correction of the effect root-knot nematode of zymotic fluid stoste
The death rate is 98.5%, and preventing positive effect after zymotic fluid dilution reduces, and nematicidal rate only has 76.3% in 10 times of dilutions,
And in 100 times of dilutions nemic death rate it is very low (<10%), the difference very little with compareing.Experiment shows zymotic fluid highly concentrated
Degree is lower to have good prevention root-knot nematode effect.
The Tham-21 bacterial strain fermentation liquor nematicidal effects of 1 various concentrations of table
| Nemic death rate | Stoste (%) | 10 times of liquid (%) | 100 times of liquid (%) | Clear water compares (%) |
| Repeat 1 | 100 | 75.5 | 12.1 | 1.8 |
| Repeat 2 | 100 | 76.9 | 8.7 | 1.4 |
| Repeat 3 | 100 | 80.9 | 11.2 | 1.3 |
| Corrected mortality | 98.5% | 76.3% | 9.4% | - |
Embodiment 5
Prevention effect of the hook-shaped Trichoderma seed coat agent processing to root-knot nematode：
After carrying out Pelleting processing using the agent of biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent to cucumber seeds according to preceding method, by seed plantation to seedling alms bowl
In.After germination, seedling occurs being inoculated with Meloidogyne incognita second instar larvae during rough leaf, every plant of inoculation 400, and
Root knot quantity was detected after 5 weeks, calculates prevention effect.Three cells, each 30 seedlings of cell, each processing are done in each processing
It is repeated 3 times.And the seed treatment not carry out biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent Cotton seeds is used as control.
Root-knot nematode severity Scaling standard is as shown in table 2.
Root knot index calculates：
Every part of expert evidence root incidence is investigated, is described according to Disease symptoms, material is investigated portionwise, records disease
Feelings rank, calculates root knot index (RKI).
Root knot index is calculated by following equation：
Root knot index (%)=∑ (s × n)/N × M × 100%
In formula：
∑ -- each state of an illness rank represents the summation of numerical value state of an illness rank diseased plant number product corresponding thereto；
S -- each state of an illness rank represents numerical value；
N -- each state of an illness rank diseased plant number；
N -- investigation total strain number；
M --- highest disease index.
Prevention effect calculation formula：
Prevention effect (%)=(1- processing root knots number/control root knot number) × 100%
Identification state of an illness partition of the level in 2 cucumber anti-Meloidogyne incognita ward of table
3rd, result of the test
Result of the test is shown in Table 3, and the root knot quantity after the processing of biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent on cucumber plant is can be seen that by experiment
Significantly reduce, 171.2/plant of root knot quantity average out in control treatment, and the root in the cucumber of biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent processing
23.1/plant of quantity average out to is tied, Tham-21 bacterial strain Pelleting kinds have reached 87.2% to the prevention effect of root-knot nematode, shown
Biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent can effectively prevent cucumber root-knot nematode.Root knot is prevented in agricultural production, particularly cucumber in greenhouse cultivation
Nematodiasis has important value.
The root knot quantity and prevention effect of 3 different disposal of table
| Treatment effect | Repeat 1 | Repeat 2 | Repeat 3 | It is average | Prevention effect % |
| Control | 169.2 | 163.2 | 181.2 | 171.2 | - |
| Pelleting seed coat agent | 27.8 | 20.1 | 21.5 | 23.1 | 87.2% |
Presently preferred embodiments of the present invention, is not intended to limit the invention, all to be made in substantive content of the present invention
Any modification, equivalent substitution and simple modifications etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of hook-shaped trichoderma strain, it is characterised in that the bacterial strain is hook-shaped trichoderma strain Tham-21, in being preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.10923.
2. biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent prepared by the hook-shaped trichoderma strain described in claim 1.
3. the application of biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent prepared by the hook-shaped trichoderma strain described in claim 2 in root-knot nematode is prevented.
4. application according to claim 3, it is characterised in that the root-knot nematode is Meloidogyne incognita and/or the north
Root-knot nematode.
5. the method that the hook-shaped trichoderma strain described in claim 1 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that this method
Comprise the following steps：
(1) hook-shaped trichoderma strain Tham-21 is inoculated in shaken cultivation in PDB culture mediums, it is 10 to obtain spore concentration7-
1010The spore suspension of/ml；
(2) the spore suspension 1-2L of step (1) and diatomite 500-700g, corn flour 200-400g, wheat bran 50-150g are mixed
After closing uniformly, solid fermentation is carried out, obtains solid fermentation thing；
(3) the solid fermentation thing of step (2) is crushed, crosses 60 mesh sieves, obtain conidia powder, conidia powder and sticker, dispersant, prevent
After rotten agent mixing, biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent is prepared.
6. the method that hook-shaped trichoderma strain according to claim 5 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that step
Suddenly PDB culture medium prescriptions are in (1)：Potato 200g/L, glucose 20g/L, surplus are water.
7. the method that hook-shaped trichoderma strain according to claim 5 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that step
Suddenly condition of culture is 25-28 DEG C in (1), shaken cultivation 6-8 days.
8. the method that hook-shaped trichoderma strain according to claim 5 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that step
Suddenly in (2), solid fermentation is carried out under the conditions of 28-30 DEG C, after fermentation 6-8 days, in 3-5 days dry under room temperature, consolidate
Body fermentate.
9. the method that hook-shaped trichoderma strain according to claim 5 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that step
Suddenly in (3), conidia powder, sticker, dispersant, the mass ratio of preservative are 85-90：1-3：8-10：1-2.
10. the method that hook-shaped trichoderma strain according to claim 5 is used to prepare biological and ecological methods to prevent plant disease, pests, and erosion seed coat agent, it is characterised in that
In step (3), sticker is sodium carboxymethylcellulose, and dispersant is sodium lignin sulfonate, and preservative is streptomysin.
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| CN106399119B (en) * | 2016-05-27 | 2019-06-11 | 浙江大学 | Biocontrol bacterial strain HZ-9 and its application in prevention and treatment soy bean cyst roundworm |
| CN105925489B (en) * | 2016-05-27 | 2019-06-11 | 浙江大学 | Biocontrol bacterial strain HZ-L9 and its application in prevention and treatment soy bean cyst roundworm |
| CN108484291A (en) * | 2018-06-08 | 2018-09-04 | 沈阳中博环保科技研发有限公司 | Discarded Cordyceps militaris rice cake bio-bacterial manure and production method |
| CN109305830B (en) * | 2018-09-10 | 2021-06-29 | 贵州省蚕业研究所 | Method for preparing pepper planting organic fertilizer from mulberry leaves and product |
| CN112063538B (en) * | 2020-09-25 | 2022-04-12 | 中国农业科学院特产研究所 | Trichoderma hamatum, microbial agent containing trichoderma hamatum and application of trichoderma hamatum |
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