CN109207381A - It is a kind of prevent and treat nematodiasis biocontrol bacterial strain SFC-3 and its application - Google Patents
It is a kind of prevent and treat nematodiasis biocontrol bacterial strain SFC-3 and its application Download PDFInfo
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Abstract
The present invention relates to the biocontrol bacterial strain SFC-3 of prevention and treatment nematodiasis and its application, can effectively solving the problems, such as the biocontrol bacterial strain of existing prevention and treatment root-knot nematode disease, that there are preventive effects is unstable, use cost is high, classification naming be trichoderma asperellum (Trichoderma asperellum), deposit number are as follows: CGMCC NO.16097, biocontrol bacterial strain SFC-3 are 1 × 10 in concentration9Under CFU/mL, there is good inhibiting effect to root-knot nematode egg hatch and second instar larvae；The bacterial strain can significantly reduce the invasion of nematode, and the control efficiency to root-knot nematode disease is 78.51%, and bacterial strain of the present invention pouring root can be used when vegetable seedling is transplanted, and have growth-promoting functions.
Description
Technical field
The present invention relates to plant protection art, in particular to a kind of biocontrol bacterial strain SFC-3 for preventing and treating nematodiasis and its application.
Background technique
Root knot nematode is a kind of soil-borne disease for seriously affecting vegetable growth, belongs to Nematoda, side tail gland guiding principle, pad
Sword mesh, different skin Superfamily, root-knot nematode section, Meloidogyne, the root of main harm crop can infect root cell, make root
Portion's conducting tissue is destroyed, to lose the ability for absorbing nutrient and moisture, causes crop malnutritive and early ageing or even whole
The withered death of strain.So far, there are about more than 80 kinds of root-knot nematode species to be reported in the world.And effective kind of China's record has
It 39, can infect including monocotyledon, dicotyledon and herbal 114 section, 3000 various plants.Cause vegetables
Root knot nematode disease mainly has following 4 kinds: Meloidogyne incognita, M hapla, javanese root knot nematode and peanut root knot
Nematode.In recent years, China's vegetable at protected field is quickly grown, and vegetable crop of the same race is planted in greenhouse year after year, causes many aged greenhouses
Root-knot nematode disease is on the rise, and there are about 50% greenhouse vegetables every year in the whole nation there are different degrees of root-knot nematode disease, causes
About more than ten00000000 yuan of economic loss.And root knot nematode post range it is wide, it is pathogenic it is strong, reproduction speed is fast, infects to have and cover
It covering property and can be propagated by sick soil, sick seedling and the modes such as pour water, prevent and treat relatively difficult, cause huge loss to vegetables production,
Therefore a very important agricultural production task is had become to the prevention and treatment of root-knot nematode.
Currently, the prevention and treatment of root knot nematode relies primarily on chemical pesticide control, but the use of chemical agent is to soil
Environment causes immeasurable destruction, while remaining pesticide is transferred to human body by food chain enrichment, finally to human body
It damages.Therefore most of insecticides tested is gradually prohibited or height limitation is used in vegetable at protected field production.Biological control is
Develop a kind of more rapid nematode control method in recent years, inhibits the generation of nematode, approach using the natural enemy of nematode
Mainly by enhancing abiogenous antagonist activities or introducing the realization of other active matters.The target that biological control is screened
Antagonist derives from natural environment, free from environmental pollution to soil environment close friend, environmentally protective, has sustainability, meets
The demand for development of green agriculture is a kind of more satisfactory Prevention Technique.The biocontrol microorganisms of existing prevention and treatment root-knot nematode disease at present
Strain haves the defects that preventive effect is unstable, use cost is high, therefore, invents the biocontrol bacterial strain gesture of prevention and treatment nematodiasis a kind of new must
Row.
Summary of the invention
For above situation, for the defect for solving the prior art, the purpose of the present invention is just to provide a kind of prevention and treatment nematodiasis
Biocontrol bacterial strain SFC-3 and its application, can effectively solve it is existing prevention and treatment root-knot nematode disease biocontrol bacterial strain there are preventive effect shakiness
Problem fixed, use cost is high.
The technical solution that the present invention solves is biocontrol bacterial strain SFC-3 of the present invention, and classification naming is trichoderma asperellum
(Trichoderma asperellum), China Committee for Culture Collection of Microorganisms was preserved on August 22nd, 2018
Common micro-organisms center, deposit number are as follows: CGMCC NO .16097.Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
The separation method of biocontrol bacterial strain SFC-3:
It weighs the serious pedotheque of 10g root-knot nematode disease to be added in the triangular flask equipped with 90mL sterile water, sets shaking table oscillation
25-35 minutes, obtaining extension rate was 10-1Soil dilution liquid, take 1mL 10-1Soil dilution liquid is added to sterile equipped with 9mL
In the test tube of water, obtaining extension rate is 10-2Soil dilution liquid, take the 300 μ L dilutions to be applied in martin substratum,
28 DEG C are cultivated 3 days, and toss edge mycelia is transferred to PDA solid medium and is purified after growing fungus colony, obtain purifying bacterial strain；
The martin substratum preparation method are as follows: 20g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0 .5g, seven water sulphur
Sour magnesium, 1% rose-bengal solution 3mL, 20g agar, addition distilled water are every in 121 DEG C of sterilizing 30min, use to 1000mL
1% streptomycin solution 0.3mL is added in 100mL culture medium；
The PDA solid medium preparation method are as follows: weigh to be cut into small pieces to be placed in water after 200g potato is peeled and boil
20g sucrose and 20g agar are added into filtrate, is settled to 1000mL with four layers of filtered through gauze by 30min, and pH value is adjusted to 7.0,
121 DEG C of sterilizing 30min.
The method for preparing biocontrol agent using biocontrol bacterial strain SFC-3 are as follows:
1) biocontrol bacterial strain SFC-3 is inoculated into PDA liquid medium in 25 DEG C on shaking table, after 180r/min cultivates 12~48h,
It is sampled in superclean bench, viable count is measured using blood counting chamber method；
2) when culture to viable count 1 × 105When CFU/mL or more, the training of PDA liquid is added according to 1:100 volume ratio in seed liquor
It supports in base, in 25 DEG C on shaking table, after 180r/min cultivates 12~48h, then 5000rpm is centrifuged 10min, every 1000mL culture medium
The wet bacterium of 10 ~ 15g of middle acquisition, every gram of wet bacterium contain 5 × 109~1011A thallus, wet bacterium and sterile water are made into biocontrol microorganisms by 1g:50mL
Agent, viable bacteria total concentration is 1 × 10 in finished product8~1010CFU/mL；
The PDA liquid medium preparation method are as follows: weigh to be cut into small pieces to be placed in water after 200g potato is peeled and boil
20g sucrose is added into filtrate, is settled to 1000mL with four layers of filtered through gauze by 30min, and pH value is adjusted to 7.0, is sterilized at 121 DEG C
30min。
The present invention is to screen the biocontrol fungi obtained from nature ring using biological control root knot nematode disease evil
It is free from environmental pollution to soil environment close friend in border, it is environmentally protective, reduce the dependence to chemical pesticide, it is nuisanceless to meet vegetables
The requirement of production, meanwhile, which has the growth for promoting vegetables, is the wound on the biocontrol bacterial strain for prevent and treat root knot nematode disease
Newly.
Specific embodiment
Below in conjunction with actual conditions, specific embodiments of the present invention will be described in further detail.
The source of biocontrol bacterial strain SFC-3 of the present invention be separated from the green house of vegetables pedotheque of Anyang City Hua County it is pure
What change obtained.
The separation method of biocontrol bacterial strain SFC-3:
1) pedotheque source: collecting soil sample root-knot nematode disease in the green house of vegetables of Anyang City Hua County is serious
Soil；
2) it weighs 10g pedotheque to be added in the triangular flask equipped with 90mL sterile water, sets shaking table and vibrate 25-35 minutes, obtain dilute
Releasing multiple is 10-1Soil dilution liquid, take 1mL 10-1Soil dilution liquid is added in the test tube equipped with 9mL sterile water, is obtained
Extension rate is 10-2Soil dilution liquid, take the 300 μ L dilutions to be applied in martin substratum, 28 DEG C are cultivated 3 days, to
It grows toss edge mycelia after fungus colony and is transferred to PDA solid medium and purified, obtain purifying bacterial strain；
The martin substratum preparation method are as follows: 20g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0 .5g, seven water sulphur
Sour magnesium, 1% rose-bengal solution 3mL, 20g agar, addition distilled water are every in 121 DEG C of sterilizing 30min, use to 1000mL
1% streptomycin solution 0.3mL is added in 100mL culture medium；
The PDA solid medium preparation method are as follows: weigh to be cut into small pieces to be placed in water after 200g potato is peeled and boil
20g sucrose and 20g agar are added into filtrate, is settled to 1000mL with four layers of filtered through gauze by 30min, and pH value is adjusted to 7.0,
121 DEG C of sterilizing 30min.
The method for being prepared into biocontrol agent using biocontrol bacterial strain SFC-3 of the present invention are as follows:
1) by the strain inoculated into PDA liquid medium in 25 DEG C on shaking table, after 180r/min cultivates 12~48h, ultra-clean
Work sampling measures viable count using blood counting chamber method；
2) when culture to viable count 1 × 105When CFU/mL or more, the training of PDA liquid is added according to 1:100 volume ratio in seed liquor
It supports in base, in 25 DEG C on shaking table, after 180r/min cultivates 12~48h, then 5000rpm is centrifuged 10min, every 1000mL culture medium
The wet bacterium of 10 ~ 15g of middle acquisition, every gram of wet bacterium contain 5 × 109~1011A thallus, wet bacterium and sterile water are made into biocontrol microorganisms by 1g:50mL
Agent, viable bacteria total concentration is 1 × 10 in finished product8~1010 CFU/mL。
Above step 1), 2) described in PDA liquid medium preparation method are as follows:
It weighs to be cut into small pieces to be placed in water after 200g potato is peeled and boils 30min, with four layers of filtered through gauze, be added into filtrate
20g sucrose is settled to 1000mL, and pH value is adjusted to 7.0, in 121 DEG C of sterilizing 30min.
The present invention is classified identification to biocontrol bacterial strain SFC-3, and is prevented the biocontrol agent using its preparation
Root knot nematode disease research is controlled, related experiment data is as follows:
One, the identification of bacterial strain
1, Microbiological Characteristics
The bacterium is cultivated on PDA solid medium, and bacterium colony initial stage is white, and center light green color, aerial hyphae is very thin, ulotrichy,
It is spread around by center, the bacterium colony back side is colourless, and bacterium colony mid-term is gradually converted into light green color, and center is dark green, aerial hyphae
It is changed into flocculence, the bacterium colony later period is integrally dark green, and flocculence mycelia is reduced, and the bacterium colony back side is colourless, conidiophore major branch
In tree-shaped, containing multistage branch, primary branch is mostly three beams alternate, and the primary branch length closer from major branch top is shorter, part
There is a stigma of fasciation on major branch top, and the top of Branches of Different Orders forms the stigma of three fasciations, stigma approximation ampoule shape, and each fraction
Branch middle part is without fasciation ampoule shape stigma, and conidium is spherical in shape, elliposoidal or oval.
2, molecular biological characteristic
Bacterial strain SFC-3 genomic DNA is extracted using CTAB method, universal primer ITS1 and ITS4 carry out PCR amplification, and PCR product is surveyed
Sequence, the website NCBI sequence alignment, the results show that the bacterial strain be trichoderma asperellum (Trichoderma asperellum).Sequence is such as
Under:
GCGGATCGACTTCACTCCAACCCAATGTGAACGTTACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGC
GTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTATTTCT
TACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAAC
GCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCC
AGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGATCGGCGTTGGGGATCGG
GACCCCTCACACGGGTGCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAA
CTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGAAATGTTGACCTCGGATCAGGTAGGA
ATACCCGCTGAACTTAACCATATCAATAAGCGGAGAAAA
Be named as trichoderma asperellum (Trichoderma asperellum), deposit number are as follows: CGMCC NO .16097.
Two, isolated test
1. the collection of root-knot nematode worm's ovum
Old complaint with root-knot nematode is cleaned, shreds root tissue with scissors, through 200 mesh and 500 mesh screen filtrations, collects 500
Mesh oversize is collected in centrifuge tube through tap water flushing, 5min is centrifuged at 2500r/min, abandons supernatant, obtain nematode worm
Ovum.Preparation concentration is 500/mL worm's ovum suspension, is saved backup in 4 DEG C of refrigerators.
2. the collection of root-knot nematode second instar larvae
Nematode worm's ovum will be obtained in above-mentioned 1, hatched in hatching apparatus, start to collect larva for 24 hours afterwards, collect one daily
It is secondary, it collects one week altogether.Preparation concentration is 250/mL larva suspension, is saved backup in 4 DEG C of refrigerators.
3. the preparation of biocontrol bacterial strain SFC-3 spore suspension
Bacterial strain SFC-3 is cultivated 5 days for 25 DEG C in PDA culture medium, mycelia is made to cover with entire plate.Bacterium is covered with aseptic water washing
The plate of filament collects spore suspension through sterile filter-cloth filtering, and adjustment spore suspension concentration is 1 × 109CFU/mL。
4. influence of the biocontrol bacterial strain SFC-3 to root-knot nematode egg hatch
Sterile 12 porocyte culture plates are taken, 3 repetitions are done in every hole inoculation 1mL spore suspension and 1mL ovum suspension, each processing.To add
Sterile water is as control.Under the conditions of temperature is 25 DEG C, respectively after 2d, 4d, 6d and 8d, the hatching situation of ovum is observed, is calculated
The hatching rate of ovum.Formula are as follows: hatching rate=hatching ovum number/total ovum number × 100%
5. biocontrol bacterial strain SFC-3 is to the lethal effect of root-knot nematode second instar larvae
Sterile 12 porocyte culture plates are taken, 3 weights are done in every hole inoculation 1mL spore suspension and 2mL second instar larvae suspension, each processing
It is multiple.To add sterile water as control.Under the conditions of temperature is 25 DEG C, respectively in 12h, for 24 hours, after 36h and 48h, observation two ages children
The lethal cases of worm calculate the lethality of second instar larvae.Formula are as follows: lethality=dead larvae number/larva sum × 100%
Three, greenhouse pot culture is tested
1. tomato seedling is cultivated
Aseptic water washing after 75% ethanol postincubation 1min is first used on tomato seeds surface, then handles 10min with 2% liquor natrii hypochloritis
Aseptic water washing afterwards, treated that seed cultivates growth in seedling-cultivation plate for surface sterilization.
2. potting soil is handled
Soil, gravel and organic matter are mixed in 1:1:0.5 ratio, in 121 DEG C of sterilizing 30min, soil is divided in diameter after sterilizing
It is stand-by in the flowerpot of 30cm.
3. root irrigation
It is 1 × 10 by concentration9The bacterium solution 50mL of CFU/mL carries out root irrigation to the root of tomato seedling, and every basin 48h is followed by root
Tie lines worm second instar larvae suspension 4mL, the concentration of second instar larvae suspension are 250/mL.
4. culture
Flowerpot random alignment after inoculation takes out tomato plant after 8 weeks in 25 DEG C of hot-house cultures, cleans up root with tap water
System measures root long, stem length and fresh root weight and fresh stem weight respectively, observes and records root knot situation, calculate preventive effect.Formula are as follows: preventive effect
(%)=(control group root knot quantity-processing group root knot number)/control group root knot number × 100%
Four, experimental result
Isolated experiment show (table 1, table 2) biocontrol bacterial strain SFC-3 concentration be 1 × 109When CFU/mL, to the worm's ovum of root-knot nematode
Hatching has good inhibiting effect, and the hatching rate of 8 days root-knot nematode eggs is only 7.24%.Biocontrol bacterial strain SFC-3 concentration be 1 ×
109When CFU/mL, there is very strong lethal effect to the second instar larvae of root-knot nematode, be to the lethality of second instar larvae after 48 hours
93.46%。
Influence of the 1 biocontrol bacterial strain SFC-3 of table to root-knot nematode egg hatch
| Egg hatching rate (%) | ||||
| Processing | 2d | 4d | 6d | 8d |
| CK | 28.73 | 49.56 | 75.87 | 97.25 |
| SFC-3 | 1.68 | 3.12 | 5.76 | 7 .24 |
Lethal effect of the 2 biocontrol bacterial strain SFC-3 of table to root-knot nematode second instar larvae
| Larva is lethal (%) | ||||
| Processing | 12h | 24h | 36h | 48h |
| CK | 0 | 0.61 | 1.23 | 1.42 |
| SFC-3 | 68.21 | 85.73 | 90.53 | 93.46 |
Greenhouse pot culture is right experiments have shown that (table 3) biocontrol bacterial strain SFC-3 can significantly reduce infects to root-knot nematode to tomato root
The preventive effect of root-knot nematode is 78.51%, and has apparent growth-promoting functions to tomato growth.
Preventive effect of the 3 biocontrol bacterial strain SFC-3 of table to tomato root-knot eelworm disease
| Processing | Root long (cm) | Stem length (cm) | Fresh root weight (g) | Fresh stem weight (g) | Root knot number | Preventive effect (%) |
| CK | 13.23 | 39.58 | 8.34 | 40.36 | 121 | — |
| SFC-3 | 18.67 | 51.72 | 12.11 | 58.82 | 26 | 78.51 |
By the above isolated test show biocontrol bacterial strain SFC-3 concentration be 1 × 109Under CFU/mL, to root-knot nematode egg hatch
There is good inhibiting effect with second instar larvae；Pot experiment shows that the microbial inoculum can significantly reduce the invasion of nematode, to root knot line
The control efficiency of parasitosis evil is 78.51%, and has the growth for promoting tomato；Bacterial strain of the present invention can be moved in vegetable seedling
Pouring root uses when cultivation, and has growth-promoting functions, and yield of vegetables 30-50% or more can be improved, and economic value improves 40% or more, while benefit
The biocontrol agent made of bacterial strain of the present invention is achieved same or similar as a result, will not enumerate in an experiment, is prevented
Tired to state, bacterial strain of the present invention is found to have actual production meaning, is suitble to large-scale promotion and application.
Claims (6)
1. a kind of biocontrol bacterial strain SFC-3, which is characterized in that its classification naming be trichoderma asperellum (Trichoderma asperellum), it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows:
CGMCC NO .16097。
2. the separation method of biocontrol bacterial strain SFC-3 described in claim 1, which is characterized in that weigh 10g root-knot nematode disease
Serious pedotheque is added in the triangular flask equipped with 90mL sterile water, sets shaking table and vibrates 25-35 minutes, obtaining extension rate is
10-1Soil dilution liquid, take 1mL 10-1Soil dilution liquid is added in the test tube equipped with 9mL sterile water, obtains extension rate
It is 10-2Soil dilution liquid, take the 300 μ L dilutions to be applied in martin substratum, 28 DEG C are cultivated 3 days, fungi to be grown
Toss edge mycelia is transferred to PDA solid medium and is purified after bacterium colony, obtains purifying bacterial strain；
The martin substratum preparation method are as follows: 20g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0 .5g, seven water sulphur
Sour magnesium, 1% rose-bengal solution 3mL, 20g agar, addition distilled water are every in 121 DEG C of sterilizing 30min, use to 1000mL
1% streptomycin solution 0.3mL is added in 100mL culture medium；
The PDA solid medium preparation method are as follows: weigh to be cut into small pieces to be placed in water after 200g potato is peeled and boil
20g sucrose and 20g agar are added into filtrate, is settled to 1000mL with four layers of filtered through gauze by 30min, and pH value is adjusted to 7.0,
121 DEG C of sterilizing 30min.
3. application of the biocontrol bacterial strain SFC-3 of any of claims 1 or 2 in preparation prevention and treatment root knot nematode disease product.
4. the method for preparing biocontrol agent using biocontrol bacterial strain SFC-3 described in as claimed in claim 1 or 22, which is characterized in that 1) will give birth to
Anti- bacterial strain SFC-3 is inoculated into PDA liquid medium in 25 DEG C on shaking table, after 180r/min cultivates 12~48h, in ultra-clean work
Platform sampling measures viable count using blood counting chamber method；
2) when culture to viable count 1 × 105When CFU/mL or more, the training of PDA liquid is added according to 1:100 volume ratio in seed liquor
It supports in base, in 25 DEG C on shaking table, after 180r/min cultivates 12~48h, then 5000rpm is centrifuged 10min, every 1000mL culture medium
The wet bacterium of 10 ~ 15g of middle acquisition, every gram of wet bacterium contain 5 × 109~1011A thallus, wet bacterium and sterile water are made into microbial inoculum by 1g:50mL, at
Viable bacteria total concentration is 1 × 10 in product8~1010CFU/mL；
The PDA liquid medium preparation method are as follows: weigh to be cut into small pieces to be placed in water after 200g potato is peeled and boil
20g sucrose is added into filtrate, is settled to 1000mL with four layers of filtered through gauze by 30min, and pH value is adjusted to 7.0, is sterilized at 121 DEG C
30min。
5. application of the biocontrol agent as claimed in claim 4 in prevention and treatment root knot nematode disease.
6. biocontrol bacterial strain SFC-3 of any of claims 1 or 2 is 1 × 10 in concentration9When CFU/mL, to root-knot nematode egg hatch
It is best with second instar larvae inhibiting effect.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817615A (en) * | 2021-11-10 | 2021-12-21 | 河南省科学院生物研究所有限责任公司 | Composite biocontrol microbial inoculum produced by fermenting Shuanghuanglian decoction dregs and application thereof |
| CN116463256A (en) * | 2023-04-04 | 2023-07-21 | 云南微态源生物科技有限公司 | Composite microbial agent and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113817615B (en) * | 2021-11-10 | 2023-05-23 | 河南省科学院生物研究所有限责任公司 | Composite biocontrol microbial agent produced by fermenting double coptis chinensis dregs and application thereof |
| CN116463256A (en) * | 2023-04-04 | 2023-07-21 | 云南微态源生物科技有限公司 | Composite microbial agent and preparation method and application thereof |
| CN116463256B (en) * | 2023-04-04 | 2023-10-20 | 云南微态源生物科技有限公司 | Composite microbial agent and preparation method and application thereof |
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