CN102242214A - Method for identifying Monilia polystroma and special primer thereof - Google Patents
Method for identifying Monilia polystroma and special primer thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying Monilia polystroma and a special primer thereof. A primer pair for identifying M. polystroma provided by the invention consists of a primer shown in a sequence 1 of a sequence table and a primer shown in a sequence 2 of a sequence table. Through the primer and the method disclosed by the invention, the M. polystroma can be quickly and exactly identified. Based on results of verification on four kinds of brown rot of kernel fruit and stone fruit, namely Monilinia fructicola, monilinia fructigena, monilinia laxa, M. polystroma, and allied species, the primer has very strong specificity. Through applying the method for identifying Monilia polystroma and the special primer thereof, the PCR (Polymerase Chain Reaction) amplification is carried out by taking extracted brown rot germ DNA as a template, conventional agarose gel electrophoresis is utilized, and the whole process can be finished within 5 hours.
Description
Technical field
The present invention relates to method and the primer special thereof of identification of M onilia polystroma.
Background technology
Brown heart (Brown Rot) be drupe and a kind of fruit, such as apple, pear, etc. production and storage period important disease.Calendar year 2001, the Georgia brown heart big area of southern US takes place, and brings direct economic loss to reach 4,300,000 dollars for the peach industry, and it is 1,500,000 dollars that the purchase sterilant brings indirect economic loss.In China, some regional sickness rate can reach 20%, and sickness rate reaches 100% when serious, has a strong impact on the edibleness of fruit and orchard worker's income.
Brown heart mainly is by Ascomycota (Ascomycete), discomycete (Discomycete), Helotiales (Helotiales), Sclerotiniaceae (Sclerotiniaceae), three kinds of pathogenic bacterias of chain sclerotinia sclerotiorum genus (Monilinia)---Monilinia fructicola, M.fructigena, M.laxa are caused.2002, van Leewen will distinguish called after Monilia polystroma from the M.fructigena bacterial strain of Japan according to morphological specificity and ITS sequence difference.M.polystroma mainly occurs in Japan, but has reported at present from Hungary and China and found this bacterium.Therefore, M.polystroma is important Quarantine Objects.M.polystroma mainly infects pomaceous fruit plant and fruits thereof such as apple, at the bacterial strain of China report from the Heilongjiang Province that adjoins with Japan, and this bacterium separates the plum from the drupe class, still unclear in different host and geographic generation and distribution situation as for this bacterium, the hazardness of M.polystroma is still waiting further investigation.Apple is the important import and export fruit of China, and along with the continuous reinforcement of various countries' fruit trade, the risk that M.polystroma propagates diffusion continues to increase.Therefore should strengthen quarantine to M.polystroma.
Four kinds of brown rot germs morphological specificity closely similar, be difficult to when lacking experience accurately judge.The Protocols in Molecular Biology that developed recently gets up has also obtained widespread use in the kind of brown rot germ is identified.In the Molecular Detection at M.polystroma; Cote (2004) (Cote; 2004, Mycologia 96:240-248) has designed the primer of identification of M .polystroma according to the random primer polymorphism; but when we test with these primers; find their often cisco unity malfunction (Fan Jinyan etc., 2007) (Fan Jinyan etc., 2007; the plant protection journal, 34:289-295).So far, the molecular method that not can be used for accurate identification of M onilia polystroma.Therefore, should be at brown rot germ from a plurality of countries and regions, the world, exploitation has the primer that stable specificity is used for identification of M onilia polystroma, sets up molecular assay method accurately, for speeding passage through customs of fruit such as apple in the international trade provide strong technical support.
Summary of the invention
An object of the present invention is to provide a kind of be used to identify and/or the primer of assistant identification Monilia polystroma right.
Provided by the present invention be used to identify and/or the primer of assistant identification Monilia polystroma right, form by the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table.
Another object of the present invention provides the method for a kind of evaluation and/or assistant identification Monilia polystroma.
The method of evaluation provided by the present invention and/or assistant identification Monilia polystroma may further comprise the steps:
Genomic dna with fungal bacterial strain to be identified is a template, to carrying out pcr amplification, obtains pcr amplification product with the described primer of claim 1; Detect described pcr amplification product,, determine that then described fungal bacterial strain to be identified is and/or the candidate is Monilia polystroma if described pcr amplification product is the fragment of 750bp.
The method of the described pcr amplification product of described detection is: agarose gel electrophoresis detects described pcr amplification product, if described pcr amplification product is shown as the band of 700bp-800bp on gel, determine that then described fungal bacterial strain to be identified is and/or the candidate is Monilia polystroma.
The method of the described pcr amplification product of described detection is: order-checking detects described pcr amplification product, if described pcr amplification product is the fragment of 750bp, determines that then described fungal bacterial strain to be identified is and/or the candidate is Monilia polystroma.
Another purpose of the present invention provides the method for a kind of evaluation and/or assistant identification brown heart pathogenic bacterium.
The method of evaluation provided by the present invention and/or assistant identification brown heart pathogenic bacterium may further comprise the steps:
Separating obtaining fungal bacterial strain to be identified from the plant tissue that brown heart takes place, is template with the genomic dna of fungal bacterial strain to be identified, to carrying out pcr amplification, obtains pcr amplification product with the described primer of claim 1; Detect described pcr amplification product, if described pcr amplification product is the fragment of 750bp, the pathogenic bacterium of then determining described brown heart comprise and/or the candidate comprises Monilia polystroma.
The method of the described pcr amplification product of described detection is: agarose gel electrophoresis detects described pcr amplification product, if described pcr amplification product is shown as the band of 700bp-800bp on gel, the pathogenic bacterium of then determining described brown heart wait and comprise and/or candidate comprises Monilia polystroma.
The method of the described pcr amplification product of described detection is: order-checking detects described pcr amplification product, if described pcr amplification product is the fragment of 750bp, the pathogenic bacterium of then determining described brown heart comprise and/or the candidate comprises Monilia polystroma.
Another purpose of the present invention provides the test kit of a kind of evaluation and/or assistant identification Monilia polystroma.
The test kit of evaluation provided by the present invention and/or assistant identification Monilia polystroma, it is right to contain the primer of being made up of the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table.
Another purpose of the present invention provides the test kit of a kind of evaluation and/or assistant identification brown heart pathogenic bacterium.
The test kit of evaluation provided by the present invention and/or assistant identification brown heart pathogenic bacterium, it is right to contain the primer of being made up of the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table.
Described primer to or described test kit identify and/or assistant identification Monilia polystroma or brown heart pathogenic bacterium in application also belong to protection scope of the present invention.
Primer provided by the invention and method can quick, accurate identification of M onilia polystroma.Authentication method of the present invention be utilize the laccase gene lcc2 of brown rot germ on drupe and a kind of fruit, such as apple, pear, etc. conservative in having kind, plant between the characteristics of variation, identify according to the section design primer that sequence difference is bigger.To being that Monilinia fructicola, M.fructigena, M.laxa, M.polystroma and allied species thereof are verified from known 4 kinds of a kind of fruit, such as apple, pear, etc.s of a plurality of countries such as China, the U.S., France, New Zealand, Britain, Italy, Japan and drupe brown rot germ, the result shows that this primer has very strong specificity.Use the present invention, the DNA that extracts brown rot germ carries out pcr amplification as template, and with conventional agarose gel electrophoresis, whole process can be finished in 5 hours.
Description of drawings
Fig. 1 is the electrophoretogram of the specific PCR amplified production of brown rot germ Monilia polystroma.
Fig. 2 is for using the electrophoretogram that Auele Specific Primer of the present invention detects the pcr amplification product of brown heart pathogenic bacterium.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Select bacterial strain for use:
Monilinia fructicola (Winter) Honey (Monilinia fructicola) is (available from the Dutch CBS167.24 of DSMZ, http://www.cbs.knaw.nl/), drupe brown rot germ (Monilinia laxa) is (available from the Dutch CBS489.50 of DSMZ, http://www.cbs.knaw.nl/), (public can obtain from China Agricultural University a kind of fruit, such as apple, pear, etc. brown rot germ (Monilinia fructigena), and the non-patent literature of putting down in writing this material is: Ioos﹠amp; Frey, 2000, European Journal of Plant Pathology 106:373-378), Monilia polystroma is (available from the Dutch CBS102686 of DSMZ, http://www.cbs.knaw.nl/), (public can obtain from China Agricultural University Botrytis cinerea (Botrytis cinerea), the non-patent literature of putting down in writing this material is: Jiang, J et al., 2009.Molecular characterization of field azoxystrobin-resistant isolates of Botrytis cinerea.Pesticide Biochemistry and Physiology 93:72-76), (public can obtain from China Agricultural University sclerotium germ (Sclerotinia sclerotiorum), the non-patent literature of putting down in writing this material is: Liu Xin et al.Pesticide Biochemistry and Physiology, 95,106-112), apple anthrax bacteria (Colletotrichum acutatum) (Zhang Rong etc., 2009, Scientia Agricultura Sinica, 42 (9): 3224-3229), Valsa mali (Valsa sp.) (available from Chinese agriculture microbial preservation administrative center ACCC30052) and Neofusicocum ribis are (available from the Dutch CBS118822 of DSMZ (http://www.cbs.knaw.nl/).
One, extracts the genomic dna of different strains
Scrape mycelia from media surface, about 25mg is put in the frozen pipe of 2ml, adds 500 μ l extraction buffers, places DNA extraction instrument FastPrep-24, and the speed of setting is 5, and the time is 40 seconds; Take out mixed solution, add 50 μ l10%SDS, 37 ℃ of water-bath 1h; Add 75 μ l 5M NaCl, with mixed solution mixing gently; Add 65 μ l CTAB/NaCl solution, fully behind the mixing in 65 ℃ of water-bath 20min; Add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), behind the abundant mixing, 10, the centrifugal 12min of 000rpm; Supernatant is transferred in another eppendorf pipe, adds the cold isopropanol of 0.6 times of volume, put upside down mixing, then 10, the centrifugal 12min of 000rpm removes supernatant; Add 500 μ l, 70% ethanol, put upside down for several times; 10, the centrifugal 12min of 000rpm removes supernatant; With the freeze concentration instrument precipitation is drained, precipitation is dissolved among the 100 μ lTE and (contains 100 μ g/ml RNase), and-20 ℃ of preservations are standby.
Two, polymerase chain reaction (PCR)
1, synthetic primer
According to the specificity of laccase2 (lcc2) gene order of Monilia polystroma, designed M.polystroma has been had specific one couple of PCR primers, the sequence of PCR primer is:
GPlaF:5 '-CCACTTCCAACATCACTC-3 ' (sequence 1 in the sequence table),
PlaR:5 '-CCCAGATTTCAAAAGCGGATTC-3 ' (sequence 2 in the sequence table).
Above-mentioned primer sequence is synthetic by match Parkson company.
2, pcr amplification reaction
The genomic dna of the different strains that obtains with above-mentioned step 1 is a template, carries out the PCR reaction with the GPlaF/PlaR primer, and each reaction includes a negative control and (promptly uses ddH
2O replaces dna profiling).
The PCR reaction system is formed: cumulative volume 25 μ l comprise 2.5 μ l, 10 * PCR Buffer (containing the rising sun hundred river companies available from Beijing), 200 μ mol/L dNTPs, each 0.2 μ mol/L of primer GPlaF and primer PlaR, 1U Taq archaeal dna polymerase, 10ng template DNA, ddH
2O supplies 25 μ l.
The PCR reaction conditions is: 95 ℃ of pre-sex change 3 minutes, and 95 ℃ of sex change 30 seconds, 64 ℃ of annealing 30 seconds, 72 ℃ were extended totally 30 circulations 1 minute; 72 ℃ were extended 10 minutes.
3, the PCR reaction product is carried out electrophoresis detection
After the PCR product 5 μ l of the different strains that above-mentioned steps 2 is obtained separate with 1.4% agarose gel electrophoresis, take pictures after dyeing with ethidium bromide (EB).
Electrophoresis detection result as shown in Figure 1, among Fig. 1, swimming lane 1 is a 1kb dna molecular amount gradient, swimming lane 2 negative contrasts, swimming lane 3 is Monilia polystroma; Swimming lane 4 is Monilinia fructicola (Winter) Honey (Monilinia fructicola); Swimming lane 5 is a kind of fruit, such as apple, pear, etc. brown rot germ (Monilinia fructigena); Swimming lane 6 is drupe brown rot germ (Monilinia laxa) bacterial strain; Swimming lane 7 is Botrytis cinerea (Botrytis cinerea); Swimming lane 8 is sclerotium germ (Sclerotinia sclerotiorum); Swimming lane 9 is apple anthrax bacteria (Colletotrichum acutatum); Swimming lane 10 is Valsa mali (Valsa sp.); Swimming lane 11 is Neofusicocum ribi.From Fig. 1 as seen, the PCR product of swimming lane 3Monilia polystroma is after the agarose gel electrophoresis colour developing, be shown as the band of 700bp-800bp (being actually 750bp) on sepharose, the pcr amplification product of other fungi and negative control is not through this big or small DNA band all occurring on the sepharose after the electrophoresis colour developing.This primer of this presentation of results is to having specificity to Monilia polystroma, and this authentication method accurately and reliably.
4, to the detection of checking order of PCR reaction product
The pcr amplification product that step 2 is obtained checks order respectively, and sequencing result shows: the amplified production of Monilia polystroma is the fragment of 750bp, and the amplified production of other fungi and negative control does not obtain the fragment of 750bp.This authentication method of this presentation of results accurately and reliably.
Strains tested:
Reference culture: select four kinds of brown rot germs as reference culture, wherein, Monilinia fructicola (Winter) Honey (Monilinia fructicola), drupe brown rot germ (Monilinia laxa), a kind of fruit, such as apple, pear, etc. brown rot germ (Monilinia fructigena) and the source of Monilia polystroma see Table 1.
Test strain: with the bacterial strain of following numbering as test strain (table 1): AST1, JX3-1, ABC8, LBD10, BMQ-3, YN1-7, BMS-4, YYH12, CLP6-1, CLA6-1, LS 13, MZ6, SL4, MW7-1, SS3 and SXP-1.
Bacterial strain uses therefor and source thereof in table 1 present embodiment
It is as follows to separate the method that obtains test strain:
Gather the sick fruit of drupe and a kind of fruit, such as apple, pear, etc. (specifically seeing Table 1) brown heart, sick belt transect is gone back to the laboratory, then, with transfering loop picking conidium on scab or the mummy, on water agar, rule, make single conidium separately, place under 22 ℃ of dark conditions and cultivated 8-16 hour.Be put under the anatomical lens after culture dish uncapped and observe, find the conidium of single sprouting and examine and confirm not have other conidium or mycelia around it, cutting the bacterium piece with scalper then is put on the PDA plate culture medium, in 22 ℃ of dark culturing about 7 days, cut the bacterium piece from colony edge and be put into the PDA slant medium and cultivated about 5 days, be placed on then preserve in 4 ℃ of refrigerators standby.
One, extracts the genomic dna of different strains
Method is with above-mentioned embodiment 1.
Two, polymerase chain reaction (PCR)
1, synthetic primer
Method is with above-mentioned embodiment 1.
2, pcr amplification reaction
Method is with above-mentioned embodiment 1.
3, the PCR reaction product is carried out electrophoresis detection
Method is with above-mentioned embodiment 1.
Electrophoresis detection result as shown in Figure 2, among Fig. 2, swimming lane 1 is a 1kb dna molecular amount gradient, swimming lane 2 negative contrasts, swimming lane 3 is Monilinia fructicola (Winter) Honey (M.fructicola), and swimming lane 4 is drupe brown rot germ (M.laxa), and swimming lane 5 is a kind of fruit, such as apple, pear, etc. brown rot germ (M.fructigena), swimming lane 6 is M.polystroma, and swimming lane 7-swimming lane 22 is the test strain of different numberings.From Fig. 2 as seen, in the reference culture, the PCR product that has only M.polystroma bacterial strain CBS102686 is after the agarose gel electrophoresis colour developing, be shown as the band of 700bp-800bp (being actually 750bp) on sepharose, and the pcr amplification product of other reference culture: M.fructicola, M.laxa and M.fructigena and negative control is not through on the sepharose this big or small DNA band appearring all after the electrophoresis colour developing; In the test strain, the PCR product of bacterial strain that is numbered LS13, MZ6, SL4, MW7-1, SS3, SXP-1 is after agarose gel electrophoresis colour developing, on sepharose, be shown as the band of 700bp-800bp (being actually 750bp), determine that these bacterial strains candidate is M.polystroma; The pcr amplification product that is numbered the bacterial strain of AST1, JX3-1, ABC8, LBD10, BMQ-3, YN1-7, BMS-4, YYH12, CLP6-1, CLA6-1 this big or small DNA band all do not occur on the sepharose after electrophoresis develops the color, determine that tentatively these bacterial strains are not M.polystroma.
4, to the detection of checking order of PCR reaction product
The pcr amplification product that step 2 is obtained checks order respectively, sequencing result shows: in the reference culture, the amplified production of M.polystroma bacterial strain CBS 102686 is the fragment of 750bp, and the amplified production of other reference culture: M.fructicola, M.laxa and M.fructigena and negative control does not obtain the fragment of 750bp; In the test strain, the amplified production that is numbered the bacterial strain of LS13, MZ6, SL4, MW7-1, SS3, SXP-1 is the fragment of 750bp, determines that these bacterial strains candidate is M.polystroma; Be numbered AST1, JX3-1, ABC8, LBD10, BMQ-3, YN1-7, BMS-4, YYH12, CLP6-1, CLA6-1 bacterial strain amplified production do not obtain the fragment of 750bp, determine that tentatively these bacterial strains are not M.polystroma.
5. further determine the kind of test strain by measuring the ITS sequence
(1) pcr amplification reaction
Cumulative volume 50 μ l comprise 5 μ l, 10 * PCR Buffer, 200 μ M dNTPs, forward and reverse primer I TS 1/ITS4 (ITS1:5 '-TCC GTA GGT GAA CCT GCG G-3 '; ITS4:5 '-TCC TCC GCT TAT TGA TAT GC-3 ') each 0.2 μ M, 2U Taq archaeal dna polymerase (containing the rising sun hundred river companies produces), the template DNA of 20ng bacterial strain SXP-1, ddH2O are supplied 50 μ l; The PCR reaction conditions is: 95 ℃ of pre-sex change 3 minutes, and 95 ℃ of sex change 30 seconds, 50 ℃ of annealing 30 seconds, 72 ℃ were extended totally 35 circulations 1.5 minutes; 72 ℃ were extended 10 minutes.
The PCR product is checked order with sequenator ABI 3730 by three rich biotech firms.
(2) sequence alignment and bacterial strain kind determines
Compare among the ITS sequence input NCBI with SXP-1, the ITS sequence Y17876 similarity of the ITS sequence of SXP-1 and Monilia polystroma is 100%, confirms that further SXP-1 is Monilia polystroma.
Claims (10)
- One kind be used to identify and/or the primer of assistant identification Monilia polystroma right, form by the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table.
- 2. identify and/or the method for assistant identification Monilia polystroma for one kind, may further comprise the steps:Genomic dna with fungal bacterial strain to be identified is a template, to carrying out pcr amplification, obtains pcr amplification product with the described primer of claim 1; Detect described pcr amplification product,, determine that then described fungi to be identified is and/or the candidate is Monilia polystroma if described pcr amplification product is the fragment of 750bp.
- 3. according to right 2 described methods, it is characterized in that:The method of the described pcr amplification product of described detection is: agarose gel electrophoresis detects described pcr amplification product, if described pcr amplification product is shown as the band of 700bp-800bp on gel, determine that then described fungal bacterial strain to be identified is and/or the candidate is Monilia polystroma.
- 4. according to right 2 described methods, it is characterized in that:The method of the described pcr amplification product of described detection is: order-checking detects described pcr amplification product, if described pcr amplification product is the fragment of 750bp, determines that then described fungal bacterial strain to be identified is and/or the candidate is Monilia polystroma.
- 5. identify and/or the method for assistant identification brown rot germ for one kind, may further comprise the steps:Separating obtaining fungal bacterial strain to be identified from the plant tissue that brown heart takes place, is template with the genomic dna of fungal bacterial strain to be identified, to carrying out pcr amplification, obtains pcr amplification product with the described primer of claim 1; Detect described pcr amplification product,, determine that then described brown rot germ comprises and/or the candidate comprises Monilia polystroma if described pcr amplification product is the fragment of 750bp.
- 6. according to right 5 described methods, it is characterized in that:The method of the described pcr amplification product of described detection is: agarose gel electrophoresis detects described pcr amplification product, if described pcr amplification product is shown as the band of 700bp-800bp on gel, determine that then described brown rot germ time comprises and/or candidate comprises Monilia polystroma.
- 7. according to right 5 described methods, it is characterized in that:The method of the described pcr amplification product of described detection is: order-checking detects described pcr amplification product, if described pcr amplification product is the fragment of 750bp, determines that then described brown rot germ comprises and/or the candidate comprises Monilia polystroma.
- 8. identify and/or the test kit of assistant identification Monilia polystroma that it is right to contain the primer of being made up of the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table for one kind.
- 9. identify and/or the test kit of assistant identification brown rot germ that it is right to contain the primer of being made up of the dna fragmentation shown in the sequence 2 in dna fragmentation shown in the sequence in the sequence table 1 and the sequence table for one kind.
- The described primer of claim 1 to, the described test kit of claim 8 or the described test kit of claim 9 identify and/or assistant identification Monilia polystroma or brown rot germ in application.
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| CN115044696A (en) * | 2022-05-07 | 2022-09-13 | 浙江大学 | Rapid early molecular detection method for vegetable sclerotiniose and application thereof |
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| CN101831492A (en) * | 2009-12-11 | 2010-09-15 | 中国农业大学 | Primer, method and kit for identifying monilinia fructicola (winter) honey |
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| CN101831492A (en) * | 2009-12-11 | 2010-09-15 | 中国农业大学 | Primer, method and kit for identifying monilinia fructicola (winter) honey |
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| Title |
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| 田利华等: "进口智利李子上核果褐腐病菌的鉴定", 《植物检疫》 * |
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| CN115044696A (en) * | 2022-05-07 | 2022-09-13 | 浙江大学 | Rapid early molecular detection method for vegetable sclerotiniose and application thereof |
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