CN105441331A - Myrothecium roridum and application thereof - Google Patents
Myrothecium roridum and application thereof Download PDFInfo
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- CN105441331A CN105441331A CN201510816351.4A CN201510816351A CN105441331A CN 105441331 A CN105441331 A CN 105441331A CN 201510816351 A CN201510816351 A CN 201510816351A CN 105441331 A CN105441331 A CN 105441331A
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Abstract
The invention relates to the technical field of biological weeding, particularly to Myrothecium roridum BC-1 with the preservation number of CGMCC No.11393. The Myrothecium roridum BC-1 is separated from blades of naturally diseased Echinochloa crusgalli (L.) Beauv. and has rapid and efficient pathogenicity for the Echinochloa crusgalli (L.) Beauv.; indoor experiments prove that conidia of the strain BC-1 are sprayed onto the blades of the Echinochloa crusgalli (L.) Beauv., scabs can appear after two days, contiguous scabs can appear on the blades of partial Echinochloa crusgalli (L.) Beauv. after seven days, the blades are withered, and the fresh weight reduction of the Echinochloa crusgalli (L.) Beauv. can be up to 93.7% after 21 days. The fungus strain, namely, the Myrothecium roridum BC-1, is safe to crops such as corn, wheat, rice and the like, has the better control effect on the Echinochloa crusgalli (L.) Beauv., has a certain biological control effect on weeds such as common crabgrass, Setaria viridis and the like, has higher potential for development of a commercial bio-herbicide, and has the very significant economic and social benefits.
Description
Technical field
The present invention relates to microbial control technical field, particularly a strain myrothecium roidium, also relates to the application of described myrothecium roidium.
Background technology
Farmland weed well developed root system, the ability absorbing moisture and nutrient is strong, fights for the conditions such as moisture, nutrient, illumination and space, the output safety of serious harm field-crop with field crops.The existing weeds Isosorbide-5-Nitrae more than 00 of China is planted, and that wherein causes material impact to crop safeties such as China's grain, vegetables has kind more than 60.According to statistics, the grain loss that China is caused by Weed infestation every year reaches 200,000,000 tons, accounts for about 11% of China's total agricultural output.But, on the other hand, in the last few years along with the research and development of efficient chemical weedicide and use, although alleviate the harm of weeds to agriculture production to a certain extent.But chemical agent continue to widely use the generation that result also in resistance weeds, the barnyard grass population of such as anti-quinclorac, the descurainia sophia (l.) webb ex prantl population of anti-nicosulfuron, the rapid generation of the resistant weeds such as the shepherd's purse population of anti-tribenuron-methyl has had a strong impact on the safety in production of China paddy rice.Therefore, low toxicity and the research and development that antagonism weeds have the biological source herbicide of same preventive effect more and more receive publicity.
Barnyard grass (Echinochloacrusgalli (L.) Beauv.) is one of widely distributed worldwide malignant weed, and one of the main weeds in Ye Shi China food crop field, causes material impact to China's grain security.At present, prevent and kill off mainly through following three kinds of approach to farmland weed: the first, prevented and kill off by artificial or mechanical effect, the method wastes time and energy, and cost is higher and effect is undesirable, and current use range is less; The second, chemical prevention, the method is simple, and cost is low, instant effect, is the main method solving Weed infestation at present.But the life-time service of chemical herbicide not only causes environmental pollution, more can cause the generation of resistance weeds, thus make the use of chemical agent be absorbed in the vicious cycle using simple dosage to increase gradually.3rd, biological control, the many collections of Biological Control of Weeds bacterial strain are from nature, and toxicity is low, and having can the feature of the repeatedly long-acting control of subculture, are to reduce the effective means that pharmaceutical chemicals uses in a large number, but Biological Control of Weeds is started late, and it is slow to take effect, and cost is higher.The microorganism resource that simultaneously can be used for carrying out weed biological control is at present less, and research concentrates in laboratory mostly, and really extensive commercial application is less.
Summary of the invention
The present invention with apple orchard infected cockspur grass for material, therefrom be separated a strain has good preventive effect myrothecium roidium bacterial strain BC-1 to barnyard grass, and further its biocontrol effect is explored, find that it is to crop safeties such as corn, wheat, paddy rice, simultaneously better to barnyard grass prevention effect, to the weeds such as lady's-grass, Herba Setariae Viridis, also there is certain biological control effect.
The present invention is obtained by following steps:
The invention provides a strain myrothecium roidium BC-1 (Myrotheciumroridum), its deposit number is CGMCCNo.11393.
Present invention also offers the conidium of myrothecium roidium BC-1.
One prepares the conidial method of myrothecium roidium BC-1:
With PDA culture medium culturing myrothecium roidium BC-1, the temperature of cultivation is 22 DEG C-28 DEG C, and the condition of cultivation is dark culturing, and the time of cultivation is 10-14 days, obtains the spore of myrothecium roidium BC-1; The temperature of described cultivation is specially 22 DEG C, 25 DEG C or 28 DEG C, and the time of described cultivation is specially 14 days.
Another object of the present invention is to provide a kind of method of cultivating myrothecium roidium BC-1CGMCCNo.11393:
With PDA culture medium culturing myrothecium roidium BC-1CGMCCNo.11393, the temperature of cultivation is 22 DEG C-28 DEG C, and the illumination condition of cultivation is dark culturing, and the time of cultivation is 10-14 days; The temperature of described cultivation is specially 22 DEG C, 25 DEG C or 28 DEG C, and the time of described cultivation is specially 10,11,12,13,14 days.
The application of conidium in biological weed control of a kind of myrothecium roidium BC-1 and/or its nutrient solution and/or myrothecium roidium BC-1.
The effective object of preferred described campelyco is barnyard grass, is secondly the weeds such as lady's-grass, Herba Setariae Viridis.
Above-mentioned campelyco is specifically made up of the conidium of myrothecium roidium BC-1, tensio-active agent and sterilized water; Containing spore 1 × 10 in preferred described campelyco
7-1 × 10
8individual/mL and tensio-active agent.
The mass concentration of preferred described tensio-active agent in described biotechnological formulation is 0.05%-0.1%.
Preferred described tensio-active agent is polysorbas20 and tween 80.
Campelyco of the present invention has strongly barnyard grass, virulence fast, the conidium of bacterial strain of the present invention is sprayed onto on the barnyard grass blade of two leaf one heart stages, namely symptom spot is there is after 2 days, after 7 days there is withered symptom in barnyard grass blade, after two weeks, the mortality ratio of barnyard grass reaches 60%, susceptible gene reaches 100%, reaches 91% after 3 weeks to the fresh weight inhibition of barnyard grass.
Biotype weedicide BC-1 of the present invention is to crop safeties such as corn, wheat, paddy rice.
Beneficial effect of the present invention:
1) myrothecium roidium BC-1 of the present invention is by the barnyard grass blade of contriver's separation from apple orchard, Jiao Miao town, Qihe County, Shandong Province natural occurrence, without any genetic modification, therefore they are discharged into occurring in nature can not cause any ecology, the hidden danger of environment, be the biological control amboceptor be perfectly safe;
2) in the present invention myrothecium roidium BC-1 to crop safeties such as corn, wheat and paddy rice, simultaneously better to barnyard grass prevention effect, to the weeds such as lady's-grass, Herba Setariae Viridis, also there is certain biological control effect, demonstrating this bacterium has larger potential development to become commercialization campelyco, has very significant economic and social benefit.
Culture presevation information
The preservation time: on October 12nd, 2015,
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number: CGMCCNo.11393,
Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101
Classification And Nomenclature: myrothecium roidium Myrotheciumroridum.
Embodiment
The present invention is described in further detail by the following examples.
Embodiment 1: the Isolation and ldentification of myrothecium roidium BC-1
The separation of 1.1 bacterial strains
The barnyard grass blade that field acquisition is naturally susceptible, with aseptic water washing 2 times, cut the disease of disease leaf, strong combining site, surface sterilization is carried out 2 minutes with 5% (v/v) chlorine bleach liquor, use 70% (v/v) alcohol surface sterilization 1 minute afterwards, with these tissues being transferred to after rinsed with sterile water in empty sterilizing culture dish, dry up in super clean bench.Leaf tissue after drying up is laid on PDA media surface, is placed in 28 DEG C of incubators and cultivates, be transferred to after growing bacterium colony in a new PDA culture dish, produce that spore is also separated through monospore, purifying obtains pure culture bacteria strain BC-1.BC-1 bacterial strain is stored in-20 DEG C of refrigerators with filter paper enzyme.
The qualification of 1.2 bacterial strains
1.2.1 identification of morphology
Method: cultivated 10 days on PDA flat board by bacterial strain BC-1, period observes and records its colonial morphology.Wash down with the conidium of sterilized water by media surface simultaneously, make conidial suspension, regulate concentration to 1 × 10
4individual spore/about ml.The conidium form of basis of microscopic observation bacterial strain BC-1; Contrast Fungal identification handbook, carries out Morphological Identification to bacterial strain BC-1.
1.2.2 Molecular Identification
CTAB method extracts the genomic dna of bacterial strain BC-1, carry out pcr amplification with primer I TS4 (5 ' TCCTCCGCTTATTGATATGC3 ') and ITS5 (5 ' GGAAGTAAAAGTCGTAACAAGG3 '), PCR primer is reclaimed and serves marine life Engineering Co., Ltd and carry out PCR primer order-checking
The GeneBank that sequence is submitted to NCBI (NationalCenterforBiotechnologyinformation, U.S.'s Bioinformatics Institute, http://www.ncbi.nlm.nih.gov) will be recorded and carry out BLAST analysis.
The preparation of 1.3 spore liquid
Myrothecium roidium BC-1 is inoculated in PDA plate culture medium, and at 28 DEG C, cultivating 14 days under dark condition, namely there are a large amount of conidiums that bacterial strain produces in media surface.Add and account for the tween 80 that total mass is 0.05%, brush the conidium of lower media surface growth by swab stick gently, collect spore liquid, individual layer lens wiping paper is crossed and is filtered unnecessary mycelia, counts and adjust conidium liquid concentration to 1 × 10 under microscope with blood counting chamber
8individual/mL.The spore suspension produced is directly used in the biological control of the weeds such as barnyard grass.
1.4BC-1 bacterial strain is to the Pathogenicity of barnyard grass
Spore suspension is prepared with reference to the method for 1.3, spray on the weeds plant such as 2 leaf phase barnyard grasses, lady's-grass and Herba Setariae Viridis, after 2 days, observe the pathogenic property of bacterial strain, there is scab after 2 days, the withered and yellow death of barnyard grass blade after spraying 7 days in the barnyard grass blade spraying BC-1 bacterial strain spore liquid.Collect over-ground part after spraying 21 days, weigh fresh weight, and add up lethality rate and fresh weight inhibition.
1.5 result
BC-1 bacterial strain was cultivated after 10 days on PDA substratum, colony diameter about 7 centimetres, and white, bacterium colony produces pitch-dark look myxospore group.Microscopic examination shows, and BC-1 bacterial strain conidium is shaft-like, and pause circle at two ends, colourless.In conjunction with ITS sequencing analysis result, show that BC-1 bacterial strain is myrothecium roidium (Myrotheciumroridum).
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 12nd, 2015 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 CGMCCNo.11393, Classification And Nomenclature is myrothecium roidium Myrotheciumroridum, and strain name is BC-1.
Embodiment 2: the mensuration of bacterial strain BC-1 host range
Prepare spore suspension with reference to the method for 1.3, and spray on 2-3 leaf phase corn, wheat and rice plant, after 7 days, observe bacterial strain to the security for examination corn, wheat and paddy rice.Within 21 days, collect crop field upper part afterwards, weigh fresh weight, and add up fresh weight inhibition.
Measure clear and definite through host range, bacterial strain BC-1 is to crop safeties such as corn, wheat and paddy rice; Have good biocontrol effect to barnyard grass, the fresh weight inhibition after two weeks is about 91%.To the weeds such as lady's-grass and Herba Setariae Viridis, there is certain biocontrol effect.
The plant that the equal right and wrong of each plant in this experiment are usually shown in and commonly use.
Concrete grammar is: plant each plant in greenhouse, at each plant strain growth to the 2-3 leaf phase, carries out spore liquid spraying to the plant of every kind of plant; Inoculate latter 21 days, carry out result statistics.
Result statistics comprise following aspect: spraying connect bacterium after the 2nd, the 7th and the 21st day, estimate each plant inoculation BC-1 after symptom and record; Wherein connect bacterium after 21 days, clip respectively processes the over-ground part of plant, takes plant fresh weight immediately, calculates fresh weight inhibition.
The calculation formula of fresh weight inhibition: fresh weight inhibition=(contrast mean fresh-process mean fresh)/contrast mean fresh × 100%.
Result is specific as follows:
Table 1BC-1 bacterial strain spore liquid compares the biological preventive effect of different weeds and crop safety
Note: in table, data are mean value ± standard error
Weeds biological and ecological methods to prevent plant disease, pests, and erosion experimental result shows, BC-1 bacterial strain spore liquid is best to barnyard grass biocontrol effect, connect bacterium after 21 days barnyard grass sickness rate be 100%, fresh weight inhibition reaches 93.7%; Poor to the biocontrol effect of lady's-grass, Herba Setariae Viridis, wheat wormwood artemisia and Amaranthus retroflexus, fresh weight inhibition is only 33.7%, 28.3% ,-3.2,16.8.
Wheat breed is selected to be: Jimai 22; Corn variety is: Shandong single 981; Rice varieties is: No. 6, Zheng's drought, above crop seed is all purchased from seeds company, carries out safety testing.
Note: in table, data are mean value ± standard error
Crop safety test-results shows, in spraying after 7 days, corn, wheat and rice leaf do not have obvious scab; Connect bacterium after 21 days, the crop fresh weights such as corn, wheat and paddy rice significantly do not suppress, and show as safety.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from spirit of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (9)
1. a strain myrothecium roidium BC-1, is characterized in that its deposit number is CGMCCNo.11393.
2. the conidium of a myrothecium roidium BC-1 according to claim 1.
3. conidium according to claim 2, it is characterized in that being obtained by following steps:
Myrothecium roidium BC-1 being inoculated in PDA plate culture medium, at 22 DEG C-28 DEG C, cultivating some skies under dark condition, namely there is the conidium that bacterial strain produces in media surface.
4. cultivate a method of myrothecium roidium BC-1, it is characterized in that comprising the following steps:
With PDA culture medium culturing myrothecium roidium BC-1, culture temperature is 22 DEG C-28 DEG C, and the illumination condition of cultivation is dark culturing, and incubation time is 10-14 days.
5. the application of conidium in campelyco of a myrothecium roidium BC-1 according to claim 1 and/or its nutrient solution or the myrothecium roidium BC-1 described in Claims 2 or 3.
6. application according to claim 5, is characterized in that the effective object of described campelyco is barnyard grass, lady's-grass, Herba Setariae Viridis or Amaranthus retroflexus.
7. the application according to claim 5 or 6, is characterized in that in described campelyco containing conidium 1 × 10
7-1 × 10
8individual/mL and tensio-active agent.
8. application according to claim 7, is characterized in that the mass concentration of described tensio-active agent in described campelyco is 0.05-0.1%.
9. application according to claim 7, is characterized in that described tensio-active agent is polysorbas20 or tween 80.
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| CN105441331B CN105441331B (en) | 2019-04-23 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886410A (en) * | 2016-04-27 | 2016-08-24 | 山东省农业科学院植物保护研究所 | Myrothecium culture medium containing barnyard grass and preparation method thereof |
| CN110904125A (en) * | 2019-11-26 | 2020-03-24 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 and application thereof |
| CN111394262A (en) * | 2020-05-14 | 2020-07-10 | 山东省农业科学院植物保护研究所 | Weed biocontrol bacterium and application thereof |
| CN112725362A (en) * | 2020-12-01 | 2021-04-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene GNAT11 and application thereof |
| CN113832036A (en) * | 2021-09-16 | 2021-12-24 | 王善仙 | Myrothecium roridum CLF007, immobilized microbial agent and application thereof |
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Patent Citations (2)
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| CN101006791A (en) * | 2006-01-26 | 2007-08-01 | 上海市农业科学院植物保护研究所 | A biocontrol strains against water hyacinth and its use |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886410A (en) * | 2016-04-27 | 2016-08-24 | 山东省农业科学院植物保护研究所 | Myrothecium culture medium containing barnyard grass and preparation method thereof |
| CN110904125A (en) * | 2019-11-26 | 2020-03-24 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 and application thereof |
| CN110904125B (en) * | 2019-11-26 | 2021-05-11 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene mfs1 and application thereof |
| CN111394262A (en) * | 2020-05-14 | 2020-07-10 | 山东省农业科学院植物保护研究所 | Weed biocontrol bacterium and application thereof |
| CN112725362A (en) * | 2020-12-01 | 2021-04-30 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene GNAT11 and application thereof |
| CN112725362B (en) * | 2020-12-01 | 2022-11-22 | 广东省微生物研究所（广东省微生物分析检测中心） | Myrothecium roridum A553 trichothecene-resistant self-protection gene GNAT11 and application thereof |
| CN113832036A (en) * | 2021-09-16 | 2021-12-24 | 王善仙 | Myrothecium roridum CLF007, immobilized microbial agent and application thereof |
| CN113832036B (en) * | 2021-09-16 | 2023-08-22 | 王善仙 | Moisture-exposed plaque bacteria CLF007, immobilized microbial agent and application thereof |
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