CN106282029B - The trichoderma harzianum strain Th-N5 of one plant of resisting carbendazim and its application - Google Patents
The trichoderma harzianum strain Th-N5 of one plant of resisting carbendazim and its application Download PDFInfo
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Abstract
The invention discloses the trichoderma harzianum strain Th-N5 of one plant of resisting carbendazim and its applications.The trichoderma harzianum strain Th-N5 is preserved in China typical culture collection center on May 6th, 2016, and deposit number is CCTCC NO:M2016251.Through infecting biological study and indoor bioassay, the bacterial strain has good preventive effect to mulberries gingko disease, and the bacterial strain has stronger drug resistance to chemical pesticide carbendazim, the carbendazim of itself and low dosage, which is used in mixed way, can thoroughly control mulberries gingko disease, greatly reduce the usage amount of chemical pesticide, solves the Pesticide Residue in sorosis.The bacterial strain is China domestic bacterial strain, not introduces from foreign countries, adapts to local natural environment.In addition, trichoderma harzianum is a kind of biocontrol fungi, as a kind of living body biological pesticide, there is the completely new mechanism of action, no pollution to the environment, the feature of noresidue different from existing chemical insecticide, be suitable for the requirement of organic foodstuff production.
Description
Technical field
The present invention relates to technical field of biological control, and in particular, to the trichoderma harzianum strain Th-N5 of one plant of resisting carbendazim
And its application of prevention and treatment mulberries gingko disease is mixed with the carbendazim of low dosage.
Background technique
Mulberries gingko disease is being commonly called as sclerotiniose, also known as Sang Bai fruit disease, belongs to fungus disease, is the main disease on sorosis
Evil, corresponding pathogen have sclerotinite (Sclerotiniasclerotiorum), mulberry reality cup cup fungi (Ciboria shiraiana), caruncula shape cup cup fungi (Ciboria carunculoides) and core ground cane bacterium
(Scleromitulashiraiana).Mulberries gingko disease infectiousness is strong, it is fast to propagate, and the state of an illness gently then influences the yield and product of mulberries
Matter, it is heavy then cause mulberry fruit can not normal mature, harvest, cause mulberries to drop in production over a large area, No kernels or seeds are gathered, as in a year of scarcity, fruit mulberry industry caused to ruin
The mulberry leaf of going out property disaster, disease carrying germ seriously threaten the production of silk cocoon, directly affect needed for mulberries secondary industry and sericultural production
Raw material sources threaten the sustainable development of sorosis industry.
Currently, the prevention and treatment of mulberries gingko disease mainly uses integrated pest control in agriculture and Techniques For Chemical Control measure, agricultural synthesis
Prevention and treatment is mainly based on reducing the pathogen radix of primary source of infection, cut off the route of transmission of pathogen, thus control efficiency is limited.
Chemical prevention mainly uses the chemical pesticides such as 50% carbendazim, 70% thiophanate methyl, 40% dimethachlon spraying, still, long-term, big
Amount ground causes pathogen drug resistance to enhance using chemical pesticides such as carbendazim, leads to chemical pesticide in sorosis product and ecological environment
Residual severely exceeds.
Trichoderma harzianum (Trichoderma harzianum) it is that fungi is controlled in the important plant disease biological and ecological methods to prevent plant disease, pests, and erosion of one kind, as
A kind of living body biological pesticide has the completely new mechanism of action, no pollution to the environment, noresidue different from existing chemical insecticide,
It can be used as the substitution protective agents of the mulberries gingko disease to develop drug resistance to chemical pesticide.But existing Kazakhstan thatch is wooden at present
Fungal strain is not still very ideal to the preventive effect of mulberries gingko disease, and most bacterial strains are to other chemical pesticides, such as more bacterium
Spirit does not have drug resistance, hinders its mixed with chemical pesticide to achieve the purpose that significantly improve mulberries gingko disease preventive effect, limit
Its application is made.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect and deficiency of existing mulberries gingko disease Prevention Technique, provide
The trichoderma harzianum strain Th-N5 of one plant of resisting carbendazim and its application, the thatch trichoderma strain Th-N5 have prevention and treatment mulberries gingko disease
Significant effect, and trichoderma harzianum strain Th-N5 has stronger drug resistance to carbendazim, mixes with the carbendazim of low dosage
Using, can thoroughly control mulberries gingkoes disease, greatly reduce the usage amount of chemical pesticide, solve the Pesticide Residue in sorosis,
Free from environmental pollution and farmland ecological environment simultaneously.
The object of the present invention is to provide one plant of trichoderma harzianum Th-N5 bacterial strain and its answering in prevention and treatment mulberries gingko disease
With.
It is a further object of the present invention to provide the bacterial strain Th-N5 to mix prevention and treatment mulberries gingko disease with the carbendazim of low dosage
Application.
Of the invention is to provide a kind of biological agent for preventing and treating mulberries gingko disease in a further object, and the preparation includes to breathe out thatch
Trichoderma Th-N5 bacterial strain.
Above-mentioned purpose of the invention is to give realization by the following technical programs.
One plant of resisting carbendazim trichoderma harzianum (Trichoderma harzianum) Th-N5 bacterial strain, the Th-N5 bacterium
Strain was preserved in China typical culture collection center (CCTCC) on May 6th, 2016, and deposit number is CCTCC No:
M2016251, classification naming number are as follows:Trichoderma harzianum Th-N5；Preservation address is Wuhan, China Wuhan University.
The trichoderma harzianum Th-N5 is isolated from mulberries gingko of the In Guangzhou Area by natural infection, then obtains through screening
Bacterial strain is purified, belongs to Deuteromycotina, Hyphomycetes, hyphomycetales, Moniliaceae by identification.
The morphological character of trichoderma harzianum Th-N5 of the invention is as follows:
Bacterium colony is deep, fine and close in PDA culture medium, and initial stage is white flock, is afterwards dirty-green or green, and the back side is in yellow
Or light green color.Conidiophore is born from the side shoot of mycelia, to raw or alternate, there is 2~3 branches, it is estranged sporogenic
Stigma is in doleiform or taper.Mycelia is very thin colourless, and tool separates, multi-branched；Conidium is mostly spherical shape, 2~2.9 × 1.9~2.6
μm, monospore is gathered into spherical on stigma.
Further, the rDNA-ITS sequence of the trichoderma harzianum Th-N5 is as shown in SEQ ID NO:1.
Trichoderma harzianum Th-N5 bacterial strain of the present invention has special efficacy in terms of preventing and treating mulberries gingko disease, and to carbendazim
There is drug resistance, can be used in mixed way with the carbendazim of low dosage, thoroughly controls mulberries gingko disease, greatly reduce the use of chemical pesticide
Amount solves the Pesticide Residue in sorosis, free from environmental pollution and farmland ecological environment.It can be used for preventing and treating the diseases such as sclerotinite simultaneously
Plurality of plant diseases caused by original.
Therefore, application of the trichoderma harzianum Th-N5 bacterial strain in prevention and treatment mulberries gingko disease, and in prevention and treatment sclerotinite
Application in terms of caused plurality of plant diseases, also all should be within protection scope of the present invention.
Meanwhile the trichoderma harzianum strain Th-N5 and carbendazim are used in mixed way the application in prevention and treatment mulberries gingko disease, with
And the application in terms of preventing and treating plurality of plant diseases caused by sclerotinite, it also all should be within protection scope of the present invention.
In addition, the present invention also provides a kind of Raw toxins extracted from trichoderma harzianum strain Th-N5.
Specifically, the extracting method of the Raw toxin is as follows: taking the spore of trichoderma harzianum Th-N5 bacterial strain, is spat with containing
The sterile water of temperature -80 is configured to spore suspension, and spore suspension is added in Czapek culture solution and is cultivated；Then add into culture solution
Enter ethyl acetate to be stripped and (extract mycotoxin therein), obtained organic phase concentration obtains Trichoderma harzianum toxin.
More specifically, the extracting method of the Raw toxin is as follows: taking the spore of trichoderma harzianum Th-N5 bacterial strain with 0.05%
Tween-80 sterile water be configured to concentration be 1 × 108Then conidium/mL spore suspension takes 2ml to be added 200ml's
In Czapek culture solution, isometric ethyl acetate is added and has bacterium by shaken cultivation 5d under the conditions of 26 ± 1 DEG C, 200rpm
Mycotoxin therein is extracted in the culture solution of silk, takes organic phase concentration to obtain Trichoderma harzianum toxin after 2 days, continuous 3 times is taken out
It is spare that the toxin of concentrate contracting is put into -20 DEG C of freezings after merging.
In addition, a kind of comprising above-mentioned trichoderma harzianum strain Th-N5 or include the anti-of the toxin extracted from the bacterial strain
Control the biological agent of mulberries gingko disease also within protection scope of the present invention.
Preferably, the biological agent of the prevention and treatment mulberries gingko disease also includes the carbendazim of 0~50 mg/L.
It is highly preferred that the carbendazim containing 1~50 mg/L and 1 × 10 in the biological agent of the prevention and treatment mulberries gingko disease5
~1 × 108Spore/ml Trichoderma harzianum Th-N5 spore suspension.
It is highly preferred that the carbendazim in the biological agent containing 25~50mg/L and 1 × 107The Kazakhstan spore/ml thatch wood
Trichoderma strain Th-N5 spore suspension.
It is highly preferred that the carbendazim in the biological agent containing 50mg/L and 1 × 107Spore/ml trichoderma harzianum
Strain Th-N5 spore suspension.
Above-mentioned biological agent answering in other plurality of plant diseases caused by prevention and treatment mulberries gingko disease and/or sclerotinite
With also should be within protection scope of the present invention.The invention has the following advantages:
Trichoderma harzianum Th-N5 disclosed by the invention is on mulberries gingko from Guangdong Province In Guangzhou Area by natural infection point
From acquisition, mulberries gingko disease can be effectively prevented.
Meanwhile the bacterial strain has stronger drug resistance to carbendazim, being used in mixed way with the carbendazim of low concentration thoroughly to control
Mulberries gingko disease, can greatly reduce the usage amount of the chemical pesticides such as carbendazim, solve the Pesticide Residue in sorosis.
In addition, the bacterial strain is China domestic bacterial strain, is not introduced from foreign countries, adapt to local natural environment.The Kazakhstan
Thatch Trichoderma is a kind of biocontrol fungi, as a kind of living body biological pesticide, is had different from the completely new of existing chemical insecticide
The mechanism of action, no pollution to the environment, the feature of noresidue, have adapted to the requirement of organic foodstuff production.
Detailed description of the invention
Fig. 1 is the colonial morphology (10 d) of trichoderma harzianum Th-N5；A: bacterium colony front；B: the bacterium colony back side.
Mycelia and the conidium form that Fig. 2 is trichoderma harzianum Th-N5.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The separation and identification of 1 trichoderma harzianum Th-N5 bacterial strain of embodiment
1. the separation of bacterial strain
(1) material: aimed strain is isolated from the mulberries gingko in the mulberries garden of In Guangzhou Area by natural infection.
(2) experiment condition
Potato dextrose agar (PDA): 200g potato+20g glucose+17~20g agar powder is poured into
In beaker and water is added makes total volume 1000ml.Through high pressure sterilization (121 DEG C, 30min).
Aseptic technique: all vessel and apparatus must be through high pressure sterilization (121 DEG C, 30min), and the operations such as inoculation are super
It is carried out in net workbench.
Condition of culture: it is placed in 26 ± 1 DEG C of illumination (14L:10D) insulating boxs and cultivates, after bacterium colony is formed, be transferred to PDA
Inclined-plane, then it is transferred to 4 DEG C of freezer storages.
(3) separation of bacterial strain
Surface sterilization is carried out to mulberries gingko sample using 5% liquor natrii hypochloritis, the sample after disinfection is in sterile water
It is put into PDA plate after washing 3 times, is placed in 26 ± 1 DEG C of insulating boxs and cultivates, newly generated bacterium colony or mycelia are transferred to new
On PDA plate, after culture observes 4-7 days bacterium colonies to be formed and produces spore, trichoderma harzianum tentatively will be judged as according to colonial morphology
Bacterium colony number be A, B, C, D, E, F totally 6 bacterial strains, then be transferred to 4 DEG C of freezer storages.
2. the Morphological Identification of bacterial strain
Identification in terms of morphology: the features such as cultural colony, mycelia, conidium and the production spore device of bacterial strain are observed.
Trichoderma harzianum bacterium colony in PDA culture medium is deep, fine and close, and initial stage is white cotton fiber shape, is afterwards dirty-green or green, back
Face is in yellow or light green color.Conidiophore is born from the side shoot of mycelia, to raw or alternate, there is 2~3 branches, it is estranged
Sporogenic stigma doleiform or taper.Mycelia is very thin colourless, and tool separates, multi-branched；Conidium be mostly it is spherical, 2~2.9 ×
1.9~2.6 μm, monospore is gathered into spherical on stigma.
3, the screening of bacterial strain
Trichoderma harzianum heredity, ecology and in terms of there is diversity, the different strains of fungi of the same race are to anti-
The pathogenicity significant difference of target is controlled, bacterial strain is different, LD50、LT50Several times to tens times can be differed.The sieve of high yield and high quality bacterial strain
Choosing is the primary premise for obtaining preferable control efficiency with acquisition.Referred to common pathogenicity, sporulation quantity, spore germination rate etc. 3
It is designated as the foundation of bacterium, screens excellent T. harzianum strains.
(1) processing of strains tested
Trichoderma harzianum A, B, C, D, E, F totally 6 separation strains obtained after purified, in PDA plate in 26 ± 1 DEG C
Insulating box (14L:10D) in culture.
(2) for trying pathogen
Sclerotinite is accessed in PDA plate, is placed in 26 ± 1 DEG C of insulating boxs and cultivates, use diameter after mycelia to be generated
Take fresh mycelia block spare for the punch of 5 mm.
(3) measurement of sporulation quantity
6 separation strains are configured to 1 × 10 respectively6Spore/ml conidiospore suspension takes 1ml suspension to instill PDA respectively
It is smoothened after culture medium, takes fresh mycelia block with the punch that diameter is 5mm after 1~2d grows mycelia, be inoculated in PDA plate
On, it is subsequently placed in culture (L:D=14:10) in incubator.5 repetitions of each bacterial strain.0.1% Tween-80 is added in 8~10d
Sterile water simultaneously collects spore, stirs 20 minutes on magnetic stirring apparatus, keeps spore fully dispersed, spore suspension is made, use blood
Ball numeration plate numeration measurement sporulation quantity.
The sporulation quantity of 6 separation strains is shown in Table 1, after growing 8 ~ 10d, between the sporulation quantity and B, C, D separation strains of separation strains A, E
Sporulation quantity significant difference, the wherein sporulation quantity highest of E separation strains.
The sporulation quantity (8 ~ 10d) of 16 separation strains of trichoderma harzianum of table
| Separation strains | Sporulation quantity (conidium/mL) |
| A | 4.51 ± 0.07×109 a |
| B | 4.07 ± 0.09×109bc |
| C | 4.16 ± 0.09×109 b |
| D | 3.69 ± 0.11×109 c |
| E | 4.75 ± 0.16×109 a |
| F | 4.27 ± 0.20×109 ab |
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
(4) measurement of spore germination rate
After 6 separation strains are cultivated 5d respectively, spore is collected with sterile water and suspension is made, sprout method examination with glass slide
It tests.It by spore suspension drop on sterilized slide glass, is placed in bottom and is covered in the culture dish of filter paper, it is sterile that 3~4 drops are added dropwise in ware
Water moisturizing (100, RH), microscopy after culture for 24 hours calculate spore germination rate (spore germination rate %=sprouting spore count/total spore count
× 100%).3 repetitions of each processing.
Conidia germination rate significant difference (see Table 2) in 6 separation strains, between E separation strains and other separation strains.Its
The spore of middle E separation strains is averaged germination rate highest, and the spore germination rate of A separation strains is minimum.
2 trichoderma harzianum of table, 6 separation strains conidia germination rates (for 24 hours)
| Separation strains | Spore quantity | Germination rate (%) M ± SE |
| A | 512 | 61.1 ± 0.2 c |
| B | 523 | 81.9 ± 2.2 ab |
| C | 611 | 84.7 ± 1.8 b |
| D | 540 | 71.6 ± 1.8 b |
| E | 613 | 89.7 ± 1.0 a |
| F | 791 | 77.0 ± 0.9 b |
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
(5) Raw toxin of trichoderma harzianum separation strains measures the bacteriostasis rate of sclerotinite
It is 1 × 10 that the spore of 6 separation strains, which is configured to concentration with 0.05% Tween-80 sterile water,8Spore/mL spore
Then sub- suspension takes 2 ml to be added in the Czapek culture solution of 200 ml, shaken cultivation under the conditions of 26 ± 1 DEG C, 200 rpm
5d；Isometric ethyl acetate is added in the culture solution with mycelia and extracts mycotoxin therein, takes organic phase dense after 2 days
Contracting obtains Trichoderma harzianum toxin, and it is spare to be put into -20 DEG C of freezings after the toxin of continuous 3 times extracting concentrations is merged.By 8 separation strains
Raw toxin dilute respectively after be added PDA culture medium in be configured to concentration be 0,5 mg/L PDA plate.Sclerotinite is inoculated into
On PDA plate, takes the mycelia block () of sclerotinite to be put into the PDA plate center with toxin after mycelia covers with plate, be placed in
It is cultivated in 26 ± 1 DEG C of insulating boxs, the 24th, 48 h survey mycelia growth diameter calculate bacteriostasis rate.Above-mentioned all test datas exist
It handles and completes on data processing software SAS system.
The results are shown in Table 3, and the bacteriostasis rate of all E separation strains significant difference, bacteriostasis rate compared with other separation strains is bright
It is aobvious to be higher than other separation strains.At the 24th, 48 h, E separation strains are most strong to the bacteriostasis rate of sclerotinite, averagely respectively 67.8 % and
The bacteriostasis rate of 64.5 %, B separation strains is lower, h) for %(24/48 51.6 %/49.9.
Bacteriostasis rate of the Raw toxin of 36 separation strains of trichoderma harzianum of table to sclerotinite
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
(6) trichoderma harzianum measures the drug resistance of carbendazim
After 6 separation strains are cultivated 5 d respectively, spore is collected with the sterile water that concentration is 5 mg/L carbendazim and is made outstanding
Liquid sprouts method test with glass slide.By spore suspension drop on sterilized slide glass, it is placed in bottom and is covered in the culture dish of filter paper,
3~4 drops sterile water moisturizing (100, RH) are added dropwise in ware, microscopy after culture for 24 hours calculates spore germination rate (spore germination rate %
=the spore count sprouted/total spore count × 100 %).3 repetitions of each processing.Carbendazim is added in PDA culture medium and is configured to
Concentration is the PDA plate of 5 mg/L.The spore of trichoderma harzianum is applied on PDA plate, Kazakhstan is taken after mycelia covers with plate
The mycelia block () of thatch Trichoderma is put into the PDA plate center that concentration is 0,5 mg/L carbendazim, is placed in 26 ± 1 DEG C of perseverances
It is cultivated in incubator, the 24th, 48h survey colony growth diameter calculate mycelial growth inhibition rate, inhibiting rate (%)=((check plot bacterium colony life
Long diameter-treatment region colony growth diameter)/check plot colony growth diameter) × 100 %.Above-mentioned all test datas are in number
It is completed according to being handled on processing software SAS system.
The results are shown in Table 4, carbendazim on the spore germination rate of E bacterial strain without influence (do not have in table 2 be added carbendazim when
Spore germination rate is 89.7 %), the difference compared with other separation strains is aobvious to B, C, E separation strains mycelial growth inhibition rate for carbendazim
It writes, carbendazim is significantly lower than other separation strains to B, C, E separation strains mycelial growth inhibition rate.The for 24 hours when carbendazim E is separated
Strain mycelial growth inhibition rate it is minimum, be 4.6 %, carbendazim is higher to A separation strains mycelial growth inhibition rate, be 13.0%(24 h).
4 trichoderma harzianum of table measures the drug resistance of carbendazim
| Separation strains | Spore germination rate (%) M ± SE(24h) | Mycelial growth inhibition rate (%) M ± SE(24h) |
| A | 57.9 ± 1.5 c | 13.0 ± 0.3 a |
| B | 72.8 ± 0.2 b | 7.8 ± 0.1 b |
| C | 79.8 ± 1.3 ab | 6.8 ± 0.1 b |
| D | 66.5 ± 3.5 bc | 11.5 ± 0.7 a |
| E | 85.6 ± 0.4 a | 4.6 ± 0.2 c |
| F | 65.0 ± 1.1 bc | 9.8 ± 0.2 ab |
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
(7) the selection result of trichoderma harzianum separation strains
When screening excellent separation strains, resisting carbendazim, pathogenic and sporulation quantity height are important reference index, spore
Germination rate takes second place.
To sum up result of study is shown, in resisting carbendazim, spore germination rate, sporulation quantity between E separation strains and other separation strains
With pathogenic etc. significant difference.The factor of various aspects is comprehensively compared, E separation strains are optimal separation bacterial strain.
4, the Molecular Identification of optimal separation bacterial strain expands the rDNA- in strain gene group using ITS1 and ITS4 as primer
Then ITS sequence carries out BLAST in GENBANK and compares analysis.
Separation strains are inoculated in Czapek culture solution, are cultivated under the conditions of 26 ± 1 DEG C, 200rpm, are harvested after 2~3 days
Mycelia, using the genomic DNA of CTAB method extracting mycelia, using the genomic DNA of bacterial strain as template, with ITS1 (sequence such as SEQ
Shown in ID NO:2) and ITS4 (sequence is as shown in SEQ ID NO:3) be the sequence fragment in the area primer amplification rDNA-ITS, and survey
BLAST is carried out after sequence compares analysis.
Wherein, PCR reaction system (25 ul): 0.25 ul of Taq enzyme, 10 x buffer, 2.5 0.2 ul of ul, dNTP,
The Primer of 10 umol/L each 1 ul, ddH219.05 ul of O, 25 ng of DNA profiling.PCR amplification program: 94 DEG C of initial denaturations
1min, 55 DEG C of 1 min of annealing, 72 DEG C of 1 min of extension, totally 30 circulations；10 min of last 72 DEG C of extensions.Amplified production is 1.5%
Ago-Gel carries out electrophoresis detection, is connected to sample presentation sequencing after pMD18-T carrier after recycling target DNA fragments.
BLAST comparison is carried out to column are sequenced, analysis shows, sequence detected and Trichoderma harzianum in GENBANKTrichoderma harzianum(Hypocrealixii) similitude of GU934533 sequence is 99.8%, illustrates to be detected
Sequence is the sequence of trichoderma harzianum, which is trichoderma harzianum, and sequence is as shown in SEQ ID NO.1.
By Molecular Identification, determine the bacterial strain be trichoderma harzianum (Trichoderma harzianum), it is sub- to belong to Fungi Imperfecti
Door, Hyphomycetes, hyphomycetales, Moniliaceae.
It is Th-N5 bacterial strain by the Strain Designation, and is preserved in China typical culture collection center on May 12nd, 2016
(CCTCC), culture presevation number is CCTCC No:M2016251, classification naming number are as follows:Trichoderma harzianum Th-
N5；Preservation address is Wuhan, China Wuhan University.
Pathogenic Tests of the Raw toxin of 2 trichoderma harzianum Th-N5 of embodiment to sclerotinite
1, bioassay be detect biocontrol fungi to the effective means of the killing extent of target pathogenic bacteria and fatality rate it
One, important reference frame can be provided for the biological control potentiality of the overall merit fungi.The present invention is with regard to the Trichoderma harzianum
The Raw toxin of bacterium Th-N5 is measured the pathogenicity of sclerotinite, to screen the optium concentration used in prevention and treatment.
Strains tested: trichoderma harzianum Th-N5 bacterial strain, sclerotinite.
(1) preparation of strains tested Raw toxin and nuclear fungal hyphae block
It takes the spore of trichoderma harzianum Th-N5 bacterial strain to be after purification configured to concentration with 0.05% Tween-80 sterile water to be
1×108Conidium/mL spore suspension, then take 2ml be added 200ml Czapek culture solution in, 26 ± 1 DEG C,
Isometric ethyl acetate is added in the culture solution with mycelia and extracts fungi therein by shaken cultivation 5d under the conditions of 200rpm
Toxin takes organic phase concentration to obtain Trichoderma harzianum toxin after 2 days, be put into -20 DEG C after the toxin of continuous 3 times extracting concentrations is merged
It freezes spare.
Sclerotinite is accessed in PDA plate, is placed in 26 ± 1 DEG C of insulating boxs and cultivates, used after mycelia covers with plate
Diameter is that the punch of 5 mm takes fresh mycelia block spare.
(2) measurement of the Raw toxin to sclerotinite bacteriostasis rate
It is added after Raw toxin is diluted in culture medium and is configured to the PDA plate that concentration is 0,2,4,8,16,32ppm.By core
The mycelia block () of cup fungi is put into the center of the PDA plate with toxin, is placed in 26 ± 1 DEG C of insulating boxs and cultivates, and the 24th, after 48h
Mycelia growth diameter is surveyed, bacteriostasis rate is calculated.Above-mentioned all test datas handle completion on data processing software SAS system.
2, result
Table 5 is the bacteriostasis rate of each treatment region Raw toxin, the results showed that, the suppression with the increase of Raw toxin concentration to sclerotinite
Bacterium rate increases, and under same concentration as time increases, weakens to the bacteriostasis rate of sclerotinite.
The Th-N5 bacterial strain Raw toxin of 5 various concentration of table measures the bacteriostasis rate of sclerotinite
| Concentration (ppm) | Bacteriostasis rate (%) M ± SE(24h) | Bacteriostasis rate (%) M ± SE(48h) |
| 1 | 19.1 ± 0.5 e | 17.4 ± 0.9 e |
| 2 | 33.3 ± 2.6 d | 31.6 ± 2.2 d |
| 4 | 53.7 ± 2.6 c | 51.9 ± 1.7 c |
| 8 | 85.2 ± 2.3 b | 82.5 ± 2.6 b |
| 16 | 100.0 ± 0.0 a | 96.9 ± 0.4 a |
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
In addition, by measurement, trichoderma harzianum Th-N5 bacterial strain to mulberry reality cup cup fungi (Ciboria shiraiana), caruncula
Shape cup cup fungi (Ciboria carunculoides) and core cane bacterium (Scleromitula shiraiana) there is preferable suppression
Bacterium effect.
3 trichoderma harzianum Th-N5 bacterial strain of embodiment mixes prevention and treatment mulberries gingko disease with low concentration carbendazim
1, method
Strains tested and carbendazim: trichoderma harzianum Th-N5 bacterial strain, carbendazim are studied purchased from the plant protection of academy of agricultural sciences, Guangdong Province
Institute.
(1) preparation of strains tested spore suspension and carbendazim mixed liquor
It is purified after obtain trichoderma harzianum Th-N5 bacterial strain, be put on PDA plate 25 ± 1 DEG C insulating box (14L:
Culture 5 days in 10D), the sterile water that 0.3% Tween-80 is added collect spore, spore suspension are stirred on magnetic stirring apparatus
20 minutes, after sporocyst is broken up uniformly, decontamination is filtered with hospital gauze, spore suspension is obtained, is remembered with blood cell counting plate
Number is made into 1 × 10 after determining spore concentration7Spore/ml spore liquid is spare.Carbendazim addition is made into 1 × 107Spore/ml
It is spare (content that carbendazim conversion is raw medicine) that the mixed liquor that concentration is 0,25,50 and 100 mg/L is configured in spore liquid.
(2) trichoderma harzianum Th-N5 mixes the prevention and treatment to mulberries gingko disease with low concentration carbendazim
Select growth and management level consistent 2 years raw potting mulberry treies be experimental subjects, mulberry tree breed be it is big by ten, by 36
Mulberry tree is randomly divided into 12 cells, and each cell 3 trees, cell surrounding have the zone of protection of 4m.
Test sets 7 processing altogether:
1) it is respectively 0(clear water),；
2) carbendazim of the mg/L/100 of 25 mg/L/50 mg/L；
3) 1 × 107Spore/ml Trichoderma harzianum spore suspension；
4) carbendazim+1 × 10 of 25 mg/L7Spore/ml Trichoderma harzianum spore suspension；
5) carbendazim+1 × 10 of 50 mg/L7Spore/ml Trichoderma harzianum spore suspension.
2 repetitions of each processing, totally 12 cells.Florescence according to a conventional method spray pesticide of each cell in mulberry tree, system
One is sprayed drug by scheme on March 1st, 2016, March 8, March 15, March 22, March 29, April 5, April 12
Onto the flower and neighbouring branch of mulberry tree.Temperature is 8~22 DEG C during preventing and treating medication, and the spray same day without rain, rains after such as spraying
It sprays again after then rain stops.The investigation of mulberries gingko disease was carried out May 10,3 branches of each tree random searching record every
The total number of fruits and the number of sick fruits of branch calculate control efficiency.Control efficiency (%)=((control group disease incidence-processing group morbidity
Rate)/control group disease incidence) × 100%.Above-mentioned all test datas handle completion on data processing software SAS system.
2, result
As shown in table 6, trichoderma harzianum Th-N5 bacterial strain is used in mixed way with low concentration carbendazim, is prevented mulberries gingko disease
Effect can reach 100%, realize the thorough control of disease, and greatly reduce the dosage of chemical pesticide carbendazim.
The control efficiency of 6 different disposal area mulberries gingko disease of table compares
| Processing | Average attack rate (%) | Control efficiency (%) |
| 0(water) | 42.2 ± 1.6 a | |
| 25 mg/L | 28.6 ± 1.8 b | 32.2 ± 1.8 c |
| 50 mg/L | 14.5 ± 0.7 c | 65.6 ± 0.7 b |
| 100 mg/L | 0.0±0.0 d | 100.0±0.0 d |
| 1×107 | 7.0 ± 1.6 d | 83.3 ± 1.5 ab |
| 25+(1×107) | 2.0 ± 0.3 d | 95.3 ± 1.4 a |
| 50+(1×107) | 0.0±0.0 d | 100.0±0.0 d |
Note: person identical with letter after column of figure indicates that difference is not significant (DMRT method) in table.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
The trichoderma harzianum strain Th-N5 of<120>one plants of resisting carbendazims and its application
<130> 2016
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 622
<212> DNA
<213> Trichoderma harzianum Th-N5
<400> 1
gttccgtagg gtgaacctgc ggagggatca ttaccgagtt tacaactccc aaacccaatg 60
tgaacgttac caaactgttg cctcggcggg atctctgccc cgggtgcgtc gcagccccgg 120
accaaggcgc ccgccggagg accaaccaaa actctttttg tataccccct cgcgggtttt 180
ttataatctg agccttctcg gcgcctctcg taggcgtttc gaaaatgaat caaaactttc 240
aacaacggat ctcttggttc tggcatcgat gaagaacgca gcgaaatgcg ataagtaatg 300
tgaattgcag aattcagtga atcatcgaat ctttgaacgc acattgcgcc cgccagtatt 360
ctggcgggca tgcctgtccg agcgtcattt caaccctcga acccctccgg ggggtcggcg 420
ttggggatcg gccctccctt agcgggtggc cgtctccgaa atacagtggc ggtctcgccg 480
cagcctctcc tgcgcagtag tttgcacact cgcatcggga gcgcggcgcg tccacagccg 540
ttaaacaccc aacttctgaa atgttgacct cggatcaggt aggaataccc gctgaactta 600
agcatatcaa taagcggagg aa 622
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tccgtagggt gaacctgcgg 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
tcctccgctt attgatatgc 20
Claims (8)
1. one plant of prevention and treatment mulberries gingko disease and the trichoderma harzianum strain Th-N5 with carbendazim resistance, which is characterized in that the bacterial strain
China typical culture collection center is preserved on May 6th, 2016, deposit number is CCTCC NO:M2016251.
2. trichoderma harzianum strain Th-N5 according to claim 1, which is characterized in that the rDNA-ITS sequence of the bacterial strain is such as
Shown in SEQ ID NO:1.
3. application of the trichoderma harzianum strain Th-N5 described in claim 1 in prevention and treatment mulberries gingko disease.
4. trichoderma harzianum strain Th-N5 described in claim 1 and carbendazim are used in mixed way the application in prevention and treatment mulberries gingko disease,
It is characterized in that, being carbendazim+1 × 10 of 50 mg/L7Spore/ml Trichoderma harzianum spore suspension.
5. a kind of biological agent for preventing and treating mulberries gingko disease, which is characterized in that include trichoderma harzianum strain described in claim 1
Th-N5。
6. biological agent according to claim 5, which is characterized in that also containing 0~50 mg/L's in the biological agent
Carbendazim.
7. biological agent according to claim 5, which is characterized in that contain the more of 1~50 mg/L in the biological agent
Bacterium spirit and 1 × 105~1 × 108Spore/ml trichoderma harzianum strain Th-N5 spore suspension.
8. application of any biological agent of claim 5~7 in prevention and treatment mulberries gingko disease.
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| Title |
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| Combination of Ethyl Acetate Extract of Trichoderma hamatum Fermentation Broth and Fungicide Carbendazim enhances Inhibition against Scleromitrula shiraiana under Laboratory Conditions;Xiaqing Chen等;《Pakistan J. Zool.》;20181005;第50卷(第6期);2239-2247 * |
| 木霉菌剂与多菌灵协同防治灰霉病试验;田连生等;《江苏农业科学》;20131231;第41卷(第12期);132-133 * |
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