CN103602677B - A kind of identify the weak pathotype of verticillium dahliae nucleic acid and primer pair and test kit - Google Patents
A kind of identify the weak pathotype of verticillium dahliae nucleic acid and primer pair and test kit Download PDFInfo
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Abstract
The invention provides a kind of nucleic acid, this nucleic acid has the sequence shown in SEQ ID NO:1.Present invention also offers the primer pair of the nucleic acid as above that increases.Present invention also offers a kind of method identifying fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae, and the method comprises the following steps: the sample to be tested of the complete genome DNA of (1) preparation containing verticillium dahliae; (2) with the complete genome DNA sample to be tested obtained in step (1) for template, use primer pair as above to carry out pcr amplification; Detect in amplified production and whether there is the nucleic acid shown in SEQ ID NO:4; (3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not the nucleic acid shown in SEQ ID No:4 in described amplified production, then indicate the bacterial strain of described verticillium dahliae to belong to high pathotype; When there is the nucleic acid shown in SEQ ID NO:4 in described amplified production, then the bacterial strain of this verticillium dahliae is indicated not belong to high pathotype.
Description
Technical field
The present invention relates to crop disease control field, particularly, relate to a kind of nucleic acid, a kind of primer pair, a kind ofly identify the method for fungal bacterial strain pathotype and a kind of test kit.
Background technology
Verticillium dahliae (Verticillium dahliae) is a kind of phytopathogenic fungi, and it can cause plant (particularly cotton) that the diseases such as verticillium occur.But, the pathotype difference of different verticillium dahliae bacterial strains is very large, such as, can cause the serious plant disease of cotton after the strain infection cotton that some pathotype is high and cause the decline of farmland output, and only causing the slight disease of cotton after the low strain infection cotton of some pathotype and less to farmland yield effect.In addition, the pathotype of verticillium dahliae also shows certain latent, and the high pathotype bacterial strain namely infected then may be not pathogenic, and seriously cause a disease on a large scale in next year.
If the main Strain type of verticillium dahliae in farmland can be detected, and its pathotype is made judge accurately, just the agricultural measure such as bound drug control and crop rotation can suppress the generation of disease, thus increase economic benefit and social benefit.
Summary of the invention
The object of the invention is the technical problem solving the pathotype how accurately judging verticillium dahliae bacterial strain, a kind of nucleic acid, a kind of primer pair are provided, a kind ofly identify the method for fungal bacterial strain pathotype and a kind of test kit.
Sequence shown in SEQ ID NO:1 is not occurring in the genome sequence of the verticillium dahliae bacterial strain of high pathotype and the sequence occurred in the genome sequence of the verticillium dahliae of low pathotype of finding of the present inventor.Thus, for the verticillium dahliae bacterial strain that a certain pathotype is to be measured, as long as identify in the genomic sequence of this verticillium dahliae bacterial strain whether there is the sequence shown in SEQ ID NO:1, can know whether this verticillium dahliae bacterial strain is high pathotype verticillium dahliae.Particularly, there is not the sequence shown in SEQ ID NO:1 if identified in the genomic sequence of this verticillium dahliae bacterial strain, then judge that this verticillium dahliae bacterial strain belongs to high pathotype; If identify in the genomic sequence of this verticillium dahliae bacterial strain and there is the sequence shown in SEQ ID NO:1, then judge that this verticillium dahliae bacterial strain does not belong to high pathotype.
To achieve these goals, on the one hand, the invention provides a kind of nucleic acid, this nucleic acid has the sequence shown in SEQ IDNO:1.
On the other hand, present invention also offers the primer pair of the nucleic acid as above that increases, this primer pair comprises the reverse primer shown in the forward primer shown in SEQ ID NO:2 and SEQ ID NO:3.
On the other hand, present invention also offers a kind of method identifying fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae (Verticillium dahliae), and the method comprises the following steps: the sample to be tested of the complete genome DNA of (1) preparation containing verticillium dahliae; (2) with the complete genome DNA sample to be tested obtained in step (1) for template, use primer pair as above to carry out pcr amplification; Detect in amplified production and whether there is the nucleic acid shown in SEQ ID NO:4; (3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not the nucleic acid shown in SEQ ID No:4 in described amplified production, then indicate the bacterial strain of described verticillium dahliae to belong to high pathotype; When there is the nucleic acid shown in SEQ ID NO:4 in described amplified production, then the bacterial strain of this verticillium dahliae is indicated not belong to high pathotype.
On the other hand, present invention also offers a kind of test kit, described test kit contains primer pair as above, and PCR reagent and/or molecular hybridization reagent.
By technique scheme, the average disease index of cotton generation verticillium that causes of the verticillium dahliae of the high pathotype that the present invention identifies is 72.1; The average disease index of cotton generation verticillium that causes of the verticillium dahliae of the low pathotype that the present invention identifies is 10.5.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
On the one hand, the invention provides a kind of nucleic acid, this nucleic acid has the sequence shown in SEQ ID NO:1.
On the other hand, present invention also offers the primer pair of the nucleic acid as above that increases, this primer pair comprises forward primer shown in SEQ ID NO:2 (5 '-GTTGATGGCTTGAAGATTGC-3 ') and the reverse primer shown in SEQ ID NO:3 (5 '-GGCTTGACTCGGAAGGATAA-3 ').
On the other hand, present invention also offers a kind of method identifying fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae (Verticillium dahliae), and the method comprises the following steps: the sample to be tested of the complete genome DNA of (1) preparation containing verticillium dahliae; (2) with the complete genome DNA sample to be tested obtained in step (1) for template, use primer pair as above to carry out pcr amplification; Detect in amplified production and whether there is the nucleic acid shown in SEQ ID NO:4; (3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not the nucleic acid shown in SEQ ID No:4 in described amplified production, then indicate the bacterial strain of described verticillium dahliae to belong to high pathotype; When there is the nucleic acid shown in SEQ ID NO:4 in described amplified production, then the bacterial strain of this verticillium dahliae is indicated not belong to high pathotype.
Wherein, the nucleic acid shown in SEQ ID NO:4 is a fragment of the nucleic acid shown in SEQ ID NO:1, it can by above-mentioned primer pair with the nucleic acid shown in SEQ ID NO:1 for template and pcr amplification obtains.
Wherein, the step preparing sample to be tested can be carried out according to the method for the preparation of the sample detecting nucleic acid of this area routine, the not special requirement of the present invention, such as can gather tissue from cotton plants, and cultivate on the substratum being organized in containing resistance gathered, and screen, distinguish and be separated the bacterium colony of verticillium dahliae, then according to (" plant genetic engineering " (what light source work, Science Press, within 2007, publish)) described in method extract the genomic dna of verticillium dahliae, can sample to be tested be prepared.
Wherein, detect in amplified production whether there is the nucleic acid shown in SEQ ID NO:4, can carry out according to the method for this area routine and standard, such as, can pass through nucleic acid electrophoresis method or real time quantitative PCR method.
On the other hand, present invention also offers a kind of test kit, described test kit contains primer pair as above, and PCR reagent and/or molecular hybridization reagent.
Below will be described the present invention by embodiment.In following examples, the bacterial strain of various verticillium dahliae belongs to the genetic resources obtained through legitimate channels.
Embodiment 1
The present embodiment is for illustration of the preparation of sample to be tested.Particularly, be the preparation of genomic dna from 80 different verticillium dahliae bacterial strains (being numbered VDG1 to VDG80).
Cut cotton plants is about 10-35cm place stem from earth's surface, be cut into 1mm
3the fritter of left and right size, be seeded in containing microbiotic (penbritin, Rifampin and Streptomycin sulphate, concentration is 100 μ g/ml) PDA substratum (containing potato 200g/L, glucose 20g/L, agar 20g/L) on, cultivate 7 days at 25 DEG C, differentiate the bacterium colony of verticillium dahliae according to the Standard Plate form of verticillium dahliae.Obtain the bacterium colony of the verticillium dahliae differentiated namely as verticillium dahliae bacterial strain sample.
According to the method described above, 80 different verticillium dahliae bacterial strains are obtained from 80 different cotton plants, by above-mentioned 80 different verticillium dahliae bacterial strains according to " plant genetic engineering " (what light source work, Science Press, 2007 publish) described in method, extract genomic dna sample respectively, obtain sample to be tested 1-80.
Embodiment 2
The present embodiment is for illustration of the detected result of the pathotype of above-mentioned 80 different verticillium dahliae bacterial strains.
Reference literature (Zhu Heqin, Feng Zili, Li Zhifang, Zhao Lihong, Shi Yongqiang. vermiculite sandy soil bottomless bowl of paper quantitatively dips in the greensickness-resistance of bacterium liquid method qualification cotton variety (being). Cotton, 2010,37 (12): 15-17) vermiculite sandy soil bottomless bowl of paper in quantitatively dips in bacterium liquid method, and the verticillium dahliae bacterial strain different to 80 described in embodiment 1 carries out cotton in seedling stage Pathotypes.The height of characterizing pathogenetic type is carried out with the disease index of described 80 different verticillium dahliae bacterial strains after the healthy cotton plants of inoculation.
Particularly, verticillium dahliae is being looked into the Bick substratum (NaNO of 2g/L
3, the K of 1g/L
2hPO
4, the MgSO of the KCl of 0.5g/L, 0.5g/L
4, the FeSO of 0.01g/L
4, the sucrose of 30g/L, the agar of 20g/L) on be cultured to generation spore, obtain spore suspension with spore under aseptic washing, and the spore concentration in spore suspension be adjusted to 5 × 10
6individual/ml.Then, sow and inoculate when the cotton seedling of paper pot has at least a slice true leaf to launch, bottom paper an official document or note, inject 10mL spore suspension, paper pot is put into paper an official document or note, inoculate more than cotton seedling 30 strain.4 weeks incidences after inoculation, investigation employing 5 grades of stagings, grade scale: 0 grade, is good for seedling, without symptom; 1 grade, 1-2 sheet cotyledon performance symptom, cotyledons turn yellow, the not aobvious symptom of true leaf; 2 grades, cotyledon and 1 true leaf performance symptom; 3 grades, 2 true leaf performance symptom; 4 grades, whole blade performance symptom, leaf abscission time serious, the top heart is withered.
According to investigation result, calculate disease index, formula is as follows:
The present embodiment detects the pathotype obtaining above-mentioned 80 different verticillium dahliae bacterial strains, by above-mentioned judging standard, identify the disease index of wherein 61 verticillium dahliae bacterial strains all higher than 35, belong to high pathotype, and the disease index of other 19 verticillium dahliae bacterial strains is all lower than 20, do not belong to high pathotype.
Above-mentioned 61 verticillium dahliae bacterial strains belonging to high pathotype are respectively: VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG68, VDG6, VDG52, VDG34, VDG7, VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, VDG17, VDG54, VDG39, VDG75, VDG20, VDG13, VDG22, VDG38, VDG19, VDG28, VDG14, VDG9, VDG53, VDG15, VDG42, VDG40 and VDG1.Above-mentioned 19 verticillium dahliae bacterial strains not belonging to high pathotype are respectively: VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51.
Embodiment 3
According to the method for regulation in the specification sheets (Solexa technology) of the sequenator Genome Analyzer of Illumina company, measure the whole genome sequence of verticillium dahliae bacterial strain (being numbered VDG1 and VDG2).
The present embodiment obtains the whole genome sequence of above-mentioned 2 different verticillium dahliae bacterial strains, by sequence alignment, identifies in the whole genome sequence of VDG1 not containing the sequence shown in SEQ ID NO:1; And containing the sequence shown in SEQ ID NO:1 in the whole genome sequence of VDG2.
By above-mentioned identical method, 61 of identifying in above-mentioned 80 different verticillium dahliae bacterial strains (are respectively VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG68, VDG6, VDG52, VDG34, VDG7, VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, VDG17, VDG54, VDG39, VDG75, VDG20, VDG13, VDG22, VDG38, VDG19, VDG28, VDG14, VDG9, VDG53, VDG15, VDG42, VDG40 and VDG1) genomic sequence in all not containing the sequence shown in SEQ ID NO:1, further, containing the sequence shown in SEQ ID NO:1 in the whole genome sequence of other 19 verticillium dahliae bacterial strains (being respectively VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51).
Result according to embodiment 2 and embodiment 3 can be determined, there is not the sequence shown in SEQ ID NO:1 if identified in the genomic sequence of verticillium dahliae bacterial strain, then this verticillium dahliae bacterial strain belongs to high pathotype; If identify in the genomic sequence of verticillium dahliae bacterial strain and there is the sequence shown in SEQ IDNO:1, then this verticillium dahliae bacterial strain does not belong to high pathotype.
Embodiment 4
Sample to be tested 1-80 obtained respectively in embodiment 1 carries out pcr amplification as template, and primer pair comprises forward primer as shown in SEQ ID NO:2 (5 '-GTTGATGGCTTGAAGATTGC-3 ') and the reverse primer shown in SEQ ID NO:3 (5 '-GGCTTGACTCGGAAGGATAA-3 ').
Concrete amplification condition comprises: 94 DEG C, 10 minutes; (94 DEG C, 30 seconds, 54 DEG C, 45 seconds, 72 DEG C, 1 minute) 35 circulations; 72 DEG C, 10 minutes, amplified production is carried out agarose gel electrophoresis detection.
Detected result illustrates: in above-mentioned 80 different verticillium dahliae bacterial strains, 61 (are respectively VDG3, VDG63, VDG61, VDG18, VDG35, VDG32, VDG73, VDG31, VDG33, VDG26, VDG55, VDG23, VDG57, VDG36, VDG11, VDG67, VDG47, VDG5, VDG29, VDG59, VDG49, VDG44, VDG21, VDG48, VDG8, VDG50, VDG25, VDG43, VDG37, VDG46, VDG16, VDG4, VDG68, VDG6, VDG52, VDG34, VDG7, VDG45, VDG12, VDG76, VDG27, VDG10, VDG41, VDG64, VDG17, VDG54, VDG39, VDG75, VDG20, VDG13, VDG22, VDG38, VDG19, VDG28, VDG14, VDG9, VDG53, VDG15, VDG42, VDG40 and VDG1) there is not the nucleic acid shown in SEQ ID NO:4, prove that these verticillium dahliae bacterial strains all do not exist the nucleic acid with the sequence shown in SEQ ID NO:1, judge that these verticillium dahliae bacterial strains belong to high pathotype accordingly.And, there is the nucleic acid shown in SEQ ID NO:4 in 19 verticillium dahliae bacterial strains (being respectively VDG70, VDG69, VDG71, VDG24, VDG79, VDG65, VDG56, VDG77, VDG74, VDG30, VDG80, VDG60, VDG2, VDG58, VDG66, VDG62, VDG72, VDG80 and VDG51) in addition, prove that these verticillium dahliae bacterial strains all exist the nucleic acid with the sequence shown in SEQ ID NO:1, judge that these verticillium dahliae bacterial strains do not belong to high pathotype accordingly.
According to the result of embodiment 2, the judged result of known embodiment 4 is entirely true, can determine thus, if identify in the genomic sequence of verticillium dahliae bacterial strain the sequence shown in specific sequence do not existed in the sequence shown in described SEQ ID NO:1, then this verticillium dahliae bacterial strain belongs to high pathotype.
Claims (1)
1. identify a method for fungal bacterial strain pathotype, described fungal bacterial strain is verticillium dahliae (Verticillium dahliae), and it is characterized in that, the method comprises the following steps:
(1) sample to be tested of the complete genome DNA of preparation containing verticillium dahliae;
(2) with the complete genome DNA sample to be tested obtained in step (1) for template, use primer pair carry out pcr amplification, this primer pair comprises the forward primer shown in SEQ ID NO:2 and the reverse primer shown in SEQ IDNO:3; Detect in amplified production and whether there is the nucleic acid shown in SEQ ID NO:4;
(3) judge the pathotype of verticillium dahliae bacterial strain according to the detected result of step (2), when there is not the nucleic acid shown in SEQ ID No:4 in described amplified production, then indicate the bacterial strain of described verticillium dahliae to belong to high pathotype; When there is the nucleic acid shown in SEQ ID NO:4 in described amplified production, then the bacterial strain of this verticillium dahliae is indicated not belong to high pathotype.
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| CN103243093A (en) * | 2012-03-01 | 2013-08-14 | 中国农业科学院作物科学研究所 | Nucleic acid, method for identifying pathotype of fungus strain and kit |
| CN103243092A (en) * | 2012-03-01 | 2013-08-14 | 中国农业科学院作物科学研究所 | Nucleic acid, method for identifying pathotype of fungus strain and kit |
| CN103255136A (en) * | 2012-03-01 | 2013-08-21 | 中国农业科学院作物科学研究所 | Nucleic acid, and method and kit used for identifying fungal strain pathogenic type |
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| CN103243093A (en) * | 2012-03-01 | 2013-08-14 | 中国农业科学院作物科学研究所 | Nucleic acid, method for identifying pathotype of fungus strain and kit |
| CN103243092A (en) * | 2012-03-01 | 2013-08-14 | 中国农业科学院作物科学研究所 | Nucleic acid, method for identifying pathotype of fungus strain and kit |
| CN103255136A (en) * | 2012-03-01 | 2013-08-21 | 中国农业科学院作物科学研究所 | Nucleic acid, and method and kit used for identifying fungal strain pathogenic type |
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