CN102154462A - Method and applications for fast detecting pathogenic bacteria molecules of bacterial soft rotting disease for banana - Google Patents
Method and applications for fast detecting pathogenic bacteria molecules of bacterial soft rotting disease for banana Download PDFInfo
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Abstract
The invention discloses a method and applications for fast detecting pathogenic bacteria molecules of bacterial soft rotting disease for banana. The method comprises the steps that: a specific primer is utilized to conduct PCR (polymerase chain reaction) on DNA contained in a sample, and the sample contains the pathogenic bacteria molecules of bacterial soft rotting disease for banana when the PCR product presents a 171bp condition. The method is simple to operate, takes less time and is large in flux. The method is used for conducting determined detection to banana seedlings, soil leach liquor of soil in a planting area, irrigation water and the like, thus realizing the purposes of construction and safety production in no disease banana seedling production base in China and simultaneously stopping of introduction of dangerous foreign disease banana.
Description
Technical field
The invention belongs to crop disease control and Plant Quarantine field, be specifically related to a kind of banana bacterial soft rot the fall ill method and the application of former bacterium molecule rapid detection, promptly utilize polymerase chain reaction (PCR) Protocols in Molecular Biology that banana bacterial soft rot evil pathogenic bacteria is carried out the highly sensitive rapid detection, can be used for the quarantine of banana seedling, the early diagnosis of field squirter and monitoring and the evaluation of germ.
Background technology
Banana bacterial soft rot pathogenic bacteria is chrysanthemum basal stem rot bacterium (Pectobacterium chrysanthemi), belongs to enterobacteriaceae (Enterobacteriaceae) erwinia (Erwinia).This pathogenic bacteria pathogenic very strong may be the strong subspecies of causing a disease.This is that dangerous disease is newly invaded by China, finds main harm dwarf banana, Brazilian any of several broadleaf plants etc. first in banana planting district, Fanyu, Guangzhou in September, 2009.This disease is systemic vascular bundle diseases, the dirty yellow in the nearly petiole of inner vanes place at first, and the petiole collapse, blade is wilted and dead.Susceptible false stem square section vascular bundle becomes green-yellow to sorrel, even black, especially all has dark-coloured gelatinoid and bacterium bacterium to overflow on the inside leaf sheath and carpopodium, false stem, rhizosphere and the single banana.Infected plant death in 6～8 days, sickness rate reaches 20～40%, and serious plot can reach more than 90%, disease plant mortality ratio 100%, and with fast speeds expansion infection area.This disease has propagates characteristics such as fast, crushing strong, causes enormous economic loss to banana production.
Banana bacterial soft rot initial stage disease symptom is similar a bit with banana blight, so the banana peasant is difficult to accurately judge cause of disease, the knowwhy that the method that traditional pathogenic bacteria separation and Culture is identified depends on the pathogenic bacteria classification of professional's rich experience and system again will affect best control opportunity like this adversely.At present, control banana bacterial soft rot does not also have efficient ways.Mainly be with and make regular check on Jiao Yuan, find that fragmentary diseased plant in time removes destruction, and spread fertilizer over the fields unslaked lime and handle the sick soil district, plant comprehensive preventive health measuress such as disease-resistant anti-sick banana variety.The morbidity under the suitable condition of humiture of banana bacterial soft rot is swift and violent, and in a single day healthy plant catches an illness can very fast death.Therefore, the banana bacterial soft rot should be strengthened emphatically the quarantine of this disease is detected to put prevention first.For preventing that the banana bacterial soft rot from importing Pest-or disease-free area into from the epidemic-stricken area, guarantee the safety in production of domestic banana, set up that a cover is stable, easy, quick, the detection of quarantining is very important the sensitive molecular detecting method to the soil of banana seedling and growing area.The PCR detection method has advantages such as highly sensitive, that precision is high, sampling amount is small, detection time is short.By this technology to qualitative detection such as banana seedling, growing area soil extraction, irrigation waters, for guaranteeing construction, the safety in production of the anosis seedling of China's banana production base, intercept simultaneously external danger in spite of illness banana to import China into significant.
Summary of the invention
Main purpose of the present invention is to overcome the shortcoming and deficiency, the method that provides a kind of banana bacterial soft rot to fall ill former bacterium molecule rapid detection of prior art.This method is easy to operation, can directly apply to production practice with the form of test kit, be used to the to carry disease germs highly sensitive rapid detection of plant tissue and soil is guaranteed for production provides healthy aseptic seedling, early warning has crucial meaning to nosophyte numerator.
Another object of the present invention is to provide the application of the method that described banana bacterial soft rot falls ill former bacterium molecule rapid detection.
Purpose of the present invention is achieved through the following technical solutions: the fall ill method of former bacterium molecule rapid detection of a kind of banana bacterial soft rot may further comprise the steps:
(1) design PCR primer, wherein:
Upstream primer LF:5 '-TTCGTCTAGAGGCCCAGGAC-3 '
Downstream primer LR:5 '-TCAGCTTGTTCCGGATTGTT-3 '
(2) sample is handled, obtained the DNA in the sample; DNA with sample is a template then, carries out the PCR reaction, and electrophoresis detection PCR product when the PCR product presents the 171bp band, promptly contains banana bacterial soft rot evil pathogenic bacteria in the sample.
Sample described in the step (2) is a banana plant when organizing, and the mode of described processing is preferably the DNA that utilizes in the CTAB method extracting banana plant tissue;
When the sample described in the step (2) was soil, the mode of described processing is preferably with sterilized water soaked soil, and the supernatant liquor that obtains is a template; More preferably per 0.5 gram soil uses the 1ml sterilized water to soak half an hour, and the supernatant liquor that obtains is a template;
When the sample described in the step (2) was disease water, the filter membrane that the mode of described processing is preferably with 0.22 μ m filtered enrichment to sick water, and the sick water of preparation greater concn is directly as template;
Reaction conditions in the reaction of PCR described in the step (2) is preferably 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min;
The fall ill method of former bacterium molecule rapid detection of described banana bacterial soft rot is applied to the quarantine of banana seedling, the early diagnosis of field squirter and monitoring and the evaluation of germ.
The present invention has following advantage and effect with respect to prior art: the present invention designs Auele Specific Primer according to the genome sequence of banana bacterial soft rot pathogenic bacteria.By this technology to qualitative detection such as banana seedling, growing area soil extraction, irrigation waters, thereby realize construction, the safety in production of the anosis seedling of China's banana production base, intercept the external danger purpose imported into of banana in spite of illness simultaneously.
Description of drawings
Fig. 1 is the method for the invention to the participate in the experiment result schematic diagram of bacterial strain DNA cloning of difference, wherein:
Swimming lane M is DNA Marker 2000; Swimming lane 1 is a banana bacterial soft rot bacterium; Swimming lane 2 is an erwinia amylovora; Swimming lane 3 is the yellow unit cell germ of bird rape; Swimming lane 4 is the Radix Dauci Sativae Erwinia; Swimming lane 5 is a banana bacterialo wilt disease germ; Swimming lane 6 is a corn bacterial blight germ; Swimming lane 7 is a bacterial leaf spot pathogenic bacteria; Swimming lane 8 is a Kidney bean wilt germ; Swimming lane 9 is the false unit cell germ of cloves; The negative contrast water of swimming lane ck-.
Fig. 2 is the detected result figure of sensitivity of the present invention, wherein:
Swimming lane M is DNA Marker 2000; Swimming lane ck+ is banana bacterial soft rot bacterium DNA; Swimming lane 1～9 is respectively 10
9, 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, the template of 10CFU/ml; The negative contrast water of swimming lane ck-.
Fig. 3 be the method for the invention to different test result of samples figure, wherein:
Swimming lane M is DNA Marker 2000; Swimming lane ck+ is banana bacterial soft rot bacterium DNA; The negative contrast water of swimming lane ck-; Swimming lane 1～4 is the DNA of ( sample 1,7,11,37 in the table 1) morbidity banana tissue; Swimming lane 5 is the banana tissue DNA of artificial inoculation pathogenic bacteria; Swimming lane 6～8 is ( sample 4,8,14 in the table 1) morbidity plot sick soil sample; Swimming lane 9 (is got 50g field soil in 160 ℃ baking oven inner drying sterilization 12h, is inoculated 10 after cooling for artificial inoculation pathogenic bacteria sick soil
9The banana bacterial soft rot bacteria suspension 5ml of CFU/ml, preparation sick soil sample) sample; Swimming lane 10 (sample 9 in the table 1) is the sick water sample in the serious plot of disease; Swimming lane 11 is the DNA of healthy banana tissue.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Round pcr detects the method for banana bacterial soft rot, the steps include:
(1) bacterial genomes DNA extraction: get frozen banana bacterial soft rot pathogenic bacteria three strains (Firstreport of a sort of banana in Mainland China Caused by a Dickeya sp. (Pectobacterium chrysanthemi) .Plant Dis.2010,94 (5): 640-640.) be inoculated into NA slant medium (extractum carnis 3g respectively, yeast extract 1g, peptone 5g, glucose 10g, agar 20g, distilled water is settled to 1000ml) go up in 35 ℃ of environment, to leave standstill and cultivate 48h, picking list colony inoculation is in liquid NA substratum, 27～35 ℃, 180rpm shaking table overnight incubation, it is stand-by to extract banana bacterial soft rot pathogenic bacteria gene group DNA respectively with bacterial genomes DNA extraction test kit (Beijing health is the century bio tech ltd).
(2) amplification banana bacterial soft rot 16S-23S rDNA ITS sequence: the bacterium general purpose I TS primer that usefulness has been reported (primer1:5 '-GAAGTCGTAACAAGG-3 '; Primer2:5 '-CAAGGCATCCACCGT-3 ') respectively three strain banana bacterial soft rot pathogenic bacteria gene group DNA is increased.Reaction system is 50 μ l:Taq MasterMix (Taq DNA polymerase, PCR Buffer, Mg
2+, the premix system formed of dNTP and PCR stablizer and toughener, concentration is 2 *) 25 μ l, each 2 μ l of the primer1 of 10 μ M and primer2, template DNA 2 μ l, RNase-Free water supplies 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min.The PCR product is identified with 1% (w/v) agarose gel electrophoresis, detect two bands that size is about 440bp and 600bp, the amplified production rubber tapping of 440bp is reclaimed, reclaiming fragment is connected with cloning vector pMD20-T Vector (the precious biotechnology of TaKaRa company limited), be converted into competent escherichia coli cell DH-5 α (the precious biotechnology of TaKaRa company limited) then, carry out blue hickie screening, after the positive bacterium colony enlarged culturing of picking, get 2 μ l bacterium liquid with the carrier universal primer (M13F:5 '-GCCAGGGTTTTCCCAGTCACGA-3 '; M13R:5 '-GAGCGGATAACAATTTCACACAGG-3 ') carries out pcr amplification, detected through gel electrophoresis, and will detect the back and confirm to contain the segmental positive colony of purpose and deliver to Shanghai Ying Jun Bioisystech Co., Ltd and carry out determined dna sequence, determined dna sequence shows that the 16S-23S rDNA ITS sequence of separating the three strain banana bacterial soft rot pathogenic bacterias that obtain is identical, as follows, and preserve positive strain.
1 GAAGTCGTAA?CAAGGTAACC?GTAGGGGAAC?CTGCGGTTGG?ATCACCTCCT?TACCGAGTAG
61 AAGTGCCTGC?GTGGTGTCCA?CACAGATTGT?CTGATGATAA?GAAAGAGTCA?AAAGCGTCTT
121?GCGAAGCTGA?CAAGTGATGT?CCCCTTCGTC?TAGAGGCCCA?GGACACCGCC?CTTTCACGGC
181?GGTAACAGGG?GTTCGAATCC?CCTAGGGGAC?GCCAATGACT?GACAGTGGGT?GAAAGGCACG
241?GTCAACGCTA?ACCTAAAACT?GATTAGAAAT?AGTCAGGTTT?ATGTTATCTG?CTCTTTAACA
301?ATCCGGAACA?AGCTGAATAA?TTTGAAACGA?CGGCATGTGA?ATGCGAATTC?ATGTGTTGTT
361?GGAGTCTCTC?AAAATACTCA?TATCCCGAGA?CACTTTCGGG?TTGTGAGGTT?AAGCGACTAA
421?GCGTACACGG?TGGATGCCTT?G
(3) design of PCR Auele Specific Primer: according to the difference of the ITS sequence of the bacterium of other kinds of download on banana bacterial soft rot pathogenic bacteria ITS sequence and the NCBI, use Premier5.0 (Canadian Premier company) design PCR special primer, upstream primer is LF:5 '-TTCGTCTAGAGGCCCAGGAC-3 ', under primer is arranged is LR:5 '-TCAGCTTGTTCCGGATTGTT-3 ', this primer amplification purpose fragment is 171bp.Primer is synthetic synthetic by Shanghai Bo Shang Bioisystech Co., Ltd.
(4) primer specificity detects: the genomic dna of step (2) the banana bacterial soft rot pathogenic bacteria gene group DNA that extracts and the pathogenic bacterium of participating in the experiment is carried out the primer specificity detection.The pathogenic bacterium of participating in the experiment have Radix Dauci Sativae Erwinia MY21 (Erwinia carotovora subsp.carotovora Ecc, the research of carrot soft rot Erwinia molecular detection technology. Agricultural University Of South China's journal, 2009,30 (3): 22-26); Xanthomonas campestris (Xanthomonas campestris, xanthomonas campestris produce matrix because of GumD clone and construction of recombinant plasmid. Food science, and 2006,27 (11): 182-184), xanthomonas campestris is the bacillary blight pathogenic bacteria of banana; Erwinia amylovora (Erwinia amylovora), banana bacterialo wilt disease bacterium (Ralstoniasolanacearum race 2), corn bacterial wilt (Pantoea stewartii subsp.StewartiiPS), bacterial leaf spot pathogenic bacteria (Xanthomonas oryze pv.oryze XO), Kidney bean wilt (Curtobacterium flaccumfaciens pv.flaccumfaciens CF), pseudomonas syringae (Pseudomonas syringae pv.syringae PSS) (foundation of banana bacterialo wilt disease bacterium real-time fluorescence PCR detection method. Tropical Agricultural University Of South China's journal, 2005,11 (1): 1-5).Wherein erwinia amylovora (Erwinia amylovora), Radix Dauci Sativae Erwinia (Erwinia carotovora subsp.carotovoraEcc) and banana bacterial soft rot bacterium belong to together not of the same racely, and xanthomonas campestris (Xanthomonascampestris), banana bacterialo wilt disease bacterium (Ralstonia solanacearum race 2) be the banana bacterial disease of reporting both at home and abroad.
Reaction system is 25 μ l:Taq MasterMix (Taq DNA polymerase, PCR Buffer, Mg
2+, the premix system formed of dNTP and PCR stablizer and toughener, concentration is 2 *) 12.5 μ l, each 1 μ l of the primer LF of 10 μ M and LR, template DNA 1 μ l, RNase-Free water supplies 25 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min.Getting 8 μ l PCR products identifies with 1% (w/v) agarose gel electrophoresis.Result (Fig. 1) shows the band amplification of having only banana bacterial soft rot bacterium (Pectobacterium chrysanthemi) that 171bp is arranged, illustrates that the detection primer LF/LR of design has good specificity.
(5) PCR detects the sensitivity of banana bacterial soft rot pathogenic bacteria: the banana bacterial soft rot pathogenic bacteria bacteria suspension of getting 32 ℃ of shaking table incubated overnight successively with 10 * gradient dilution up to 10
9*, then, 10
4*, 10
5*, 10
6*, 10
7*, 10
8*, 10
9* diluent is respectively got 100 μ l spread plates, and each concentration gradient is done three repetitions.Flat board calculates colony growth number under each concentration gradient after cultivating 48 hours under 32 ℃ of conditions.Find 10
6210 bacterium colonies of dull and stereotyped average growth of * coating, 10
7* 20 bacterium colonies, 10 are arranged
5* can't count, can judge that thus original bacteria liquid concentration is 2 * 10
9CFU/ml.With 10
99 dilution gradients of CFU/ml～10CFU/ml are that template is done the PCR detection.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min.Reaction system is 25 μ l:Taq MasterMix (Taq DNA polymerase, PCR Buffer, Mg
2+, the premix system formed of dNTP and PCR stablizer and toughener, concentration is 2 *) 12.5 μ l, each 1 μ l of the primer LF of 10 μ M and LR, template DNA 2 μ l, Rnase-Free water supplies 25 μ l.Get 8 μ l PCR products after reaction finishes and identify that with 1% (w/v) agarose gel electrophoresis result (Fig. 2) shows 10
9～10
3The CFU/ml template all has corresponding amplified band, 10
2～10CFU/ml is amplification not, and this explanation can detected concentration dilution point of accumulation be 10
3CFU/ml, promptly 4 bacteriums (the bacteria suspension original content is 2 * 10
9CFU/ml).This detection primer of this presentation of results has than higher detection sensitivity banana bacterial soft rot bacterium pathogenic bacteria.
(6) application of PCR detection method: when being used to detect banana plant and organizing the banana bacterial soft rot, extract banana plant tissue DNA to be detected with the CTAB method, in detecting soil during banana bacterial soft rot bacterium, a small amount of (0.5g) pedotheque is put into the 1ml sterilized water soak half an hour, the centrifugal 10min of 12000r/min, supernatant is as template, in detecting sick water during banana bacterial soft rot bacterium, filter membrane with 0.22 μ m filters enrichment to sick water, and the sick water of preparation greater concn is directly as template; Carrying out PCR according to following reaction conditions and system detects: reaction conditions is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min.Reaction system is 25 μ l:TaqMasterMix (Taq DNA polymerase, PCR Buffer, Mg
2+, the premix system formed of dNTP and PCR stablizer and toughener, concentration is 2 *) 12.5 μ l, each 1 μ l of the primer LF of 10 μ M and LR, template DNA 2 μ l, RNase-Free water supplies 25 μ l.Getting 8 μ l PCR products after reaction finishes identifies with 1% (w/v) agarose gel electrophoresis.
(1) sample collecting and pathogenic bacteria unit cell isolation identification
Banana planting ground (as shown in table 1) such as GuangZhou, Guangdong Province in September, 2010 city's Fanyu District and Nansha District, Dongguan City, Zhongshan city, Zhuhai City, Gaozhou City, Huazhou City, East Sea Island, Zhanjiang City and Xuwen County, Nanning city, Zhangzhou City, Fujian Province, Dengmai County, Hainan Province, Lingao County, Le Dong county, Dongfang Shi, gather the diseased plant sample, lesion soil sample and water sample are taken back and the bacterial isolate bacterium.
(2) the banana bacterial soft rot detects:
1. the processing of sample: the CTAB method is extracted the false stem tissue gene of diseased plant group DNA; Every 0.5g soil sample directly is soaked in 1ml sterilized water half hour, the centrifugal 5min of 5000rpm, and it is stand-by to get supernatant; Water sample need not to handle.
2. PCR reaction:
In the 25 μ l reaction systems:
Taq?MasterMix 12.5μl
Upstream primer LF (10 μ M) 1 μ l
Downstream primer LR (10 μ M) 1 μ l
RNase-Free water supplies 25 μ l.
The PCR reaction conditions is:
95 ℃ of pre-sex change 5min;
94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations;
72 ℃ are extended 10min.
3. the PCR product is identified with 1% (w/v) agarose gel electrophoresis, analyzes with gel imaging system behind the electrophoresis.
(3) detected result is as shown in table 1, detects 50 fens samples altogether, and only 4 parts of detected results are negative, and being detected as power is 92%.The part electrophoresis result as shown in Figure 3, the positive of diseased plant sample, lesion soil sample, water sample amplification all has specific band at the 171bp place.
(4) the banana bacterial soft rot bacterium unit cell that separation is obtained carries out being diluted to 10 after the incubated overnight according to the method for above-mentioned culturing bacterium
6CFU/ml carries out virulence mensuration with the syringe tieback on the banana seedlings of health, check incidence behind the 3-5d, and all cause that the unit cell of plant morbidity is all consistent with the positive findings that PCR detects.
By banana bacterial soft rot disease survey, Dongfang Shi, Hainan Province, Li Autonomous County of ledong, GuangZhou, Guangdong Province city, Dongguan City, Zhongshan city, Zhuhai City, the morbidity of Zhangzhou City, Fujian Province is heavier, serious plot sickness rate is up to 80%, take second place in Lingao County, Hainan Province, Zhanjiang City, Guangdong Province, Dengmai County, Hainan and Guangdong Maoming City are light slightly, and there is fragmentary discovery in portion part plot.
The detected result and the banana bacterial soft rot evil investigation result basically identical that utilize the present invention to obtain, this shows that the method for the invention has higher feasibility, and can carry out special detection to incidence tissue, sick soil, sick water.To detecting the male plot, remind the banana peasant when extracing Lao Ye, slicing off operation such as the farming of inhaling bud, at first sharpener was carried out disinfection 3-5 minute, in case the cross infection between diseased plant and healthy plant of banana bacterial soft rot.Can be used for simultaneously the construction in the anosis seedling of China's banana base, the customs quarantine control port has good practicality to the detection of banana bacterial soft rot bacterium etc.
Table 1 banana bacterial soft rot collected specimens detected result
| Sample number into spectrum | The sample title | Sample source | Acquisition time | Collecting location | Detected result |
| 1 | Dg-1 | The false stem of diseased plant | 2010.09.08 | The Dongguan | + |
| 2 | Dg-2 | The false stem of diseased plant | 2010.09.08 | The Dongguan | + |
| 3 | Dg-3 | The false stem of diseased plant | 2010.09.08 | The Dongguan | + |
| 4 | Dg-4 | Sick soil | 2010.09.08 | The Dongguan | + |
| 5 | Dg-5 | Sick water | 2010.09.08 | The Dongguan | - |
| 6 | Py-1 | The false stem of diseased plant | 2010.09.15 | The Guangzhou Fanyu District | + |
| 7 | Py-2 | The false stem of diseased plant | 2010.09.15 | The Guangzhou Fanyu District | + |
| 8 | Py-3 | Sick soil | 2010.09.15 | The Guangzhou Fanyu District | + |
| 9 | Py-4 | Sick water | 2010.09.15 | The Guangzhou Fanyu District | + |
| 10 | Ns-1 | The false stem of diseased plant | 2010.09.22 | The Guangzhou Nansha District | + |
| 11 | Ns-2 | The false stem of diseased plant | 2010.09.22 | The Guangzhou Nansha District | + |
| 12 | Ns-3 | The false stem of diseased plant | 2010.09.22 | The Guangzhou Nansha District | - |
| 13 | Ns-4 | The false stem of diseased plant | 2010.09.22 | The Guangzhou Nansha District | + |
| 14 | Ns-5 | Sick soil | 2010.09.22 | The Guangzhou Nansha District | + |
| 15 | Zs-1 | The false stem of diseased plant | 2010.09.28 | The Zhongshan city | + |
| 16 | Zs-2 | The false stem of diseased plant | 2010.09.28 | The Zhongshan city | + |
| 17 | Zh-1 | The false stem of diseased plant | 2010.10.08 | The Zhuhai City | + |
| 18 | Zh-2 | The false stem of diseased plant | 2010.10.08 | The Zhuhai City | + |
| 19 | Zh-3 | The false stem of diseased plant | 2010.10.08 | The Zhuhai City | + |
| 20 | Gz-1 | The false stem of diseased plant | 2010.10.28 | Gaozhou county, Zhanjiang | + |
| 21 | Gz-2 | The false stem of diseased plant | 2010.10.28 | Gaozhou county, Zhanjiang | + |
| 22 | Gz-3 | The false stem of diseased plant | 2010.10.28 | Gaozhou county, Zhanjiang | + |
| 23 | Gz-4 | The false stem of diseased plant | 2010.10.28 | Gaozhou county, Zhanjiang | + |
| 24 | Gz-5 | The false stem of diseased plant | 2010.10.28 | Gaozhou county, Zhanjiang | + |
| 25 | Nz-1 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 26 | Nz-2 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 27 | Nz-3 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 28 | Nz-4 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 29 | Nz-5 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 30 | Nz-6 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 31 | Nz-7 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | - |
| 32 | Nz-8 | The false stem of diseased plant | 2010.10.28 | Island in Guangdong Province, Zhanjiang | + |
| 33 | Xw-1 | The false stem of diseased plant | 2010.10.28 | Xuwen, Zhanjiang | + |
| 34 | Xw-2 | The false stem of diseased plant | 2010.10.28 | Xuwen, Zhanjiang | + |
| 35 | Xw-3 | The false stem of diseased plant | 2010.10.28 | Xuwen, Zhanjiang | + |
| 36 | Ct-1 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 37 | Ct-2 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 38 | Ct-3 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 39 | Ct-4 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 40 | Ct-5 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | - |
| 41 | Ct-6 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 42 | Ct-7 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 43 | Nj-1 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 44 | Nj-2 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 45 | Nj-3 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 46 | Nj-4 | The false stem of diseased plant | 2010.10.20 | The Zhangzhou City, Fujian | + |
| 47 | GX-1 | The false stem of diseased plant | 2010.11.15 | The Nanning | + |
| 48 | GX-2 | The false stem of diseased plant | 2010.11.15 | The Nanning | + |
| 49 | GX-3 | The false stem of diseased plant | 2010.11.15 | The Nanning | + |
| 50 | GX-4 | The false stem of diseased plant | 2010.11.15 | The Nanning | + |
Annotate :+: strong positive;-: feminine gender
The present invention does not carry out above-mentioned detection to having the banana tissue and the pedotheque that infect the banana bacterial soft rot, does not find that molecular weight is that the 171bp place has the specific band amplification.
Can learn from above result, use the primer LF/LR in the ITS sequence specific zone of banana bacterial soft rot, banana plant tissue and pedotheque to supply examination detect, if can amplify the fragment of 171bp specifically, can judge to have had banana bacterial soft rot bacterium in banana tissue or the pedotheque.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
- The method of former bacterium molecule rapid detection 1.-kind banana bacterial soft rot is fallen ill is characterized in that may further comprise the steps:(1) design PCR primer, wherein:Upstream primer LF:5 '-TTCGTCTAGAGGCCCAGGAC-3 'Downstream primer LR:5 '-TCAGCTTGTTCCGGATTGTT-3 ';(2) sample is handled, obtained the DNA in the sample; DNA with sample is a template then, carries out the PCR reaction, and electrophoresis detection PCR product when the PCR product presents the 171bp band, promptly contains banana bacterial soft rot evil pathogenic bacteria in the sample.
- 2. according to the fall ill method of former bacterium molecule rapid detection of the described banana bacterial soft rot of claim 1, it is characterized in that: the sample described in the step (2) is a banana plant when organizing, and the mode of described processing is the DNA that utilizes in the CTAB method extracting banana plant tissue.
- 3. according to the fall ill method of former bacterium molecule rapid detection of the described banana bacterial soft rot of claim 1, it is characterized in that: when the sample described in the step (2) was soil, the mode of described processing was for to soak soil with sterilized water, and the supernatant liquor that obtains is a template.
- 4. according to the fall ill method of former bacterium molecule rapid detection of the described banana bacterial soft rot of claim 3, it is characterized in that: the mode of described processing is that per 0.5 gram soil uses the 1ml sterilized water to soak half an hour, and the supernatant liquor that obtains is a template.
- 5. according to the fall ill method of former bacterium molecule rapid detection of the described banana bacterial soft rot of claim 1, it is characterized in that: when the sample described in the step (2) is disease water, the mode of described processing is filtered enrichment for the filter membrane with 0.22 μ m to sick water, prepares the higher sick water of germ concentration as template.
- 6. according to the fall ill method of former bacterium molecule rapid detection of the described banana bacterial soft rot of claim 1, it is characterized in that: the reaction conditions in the reaction of PCR described in the step (2) is 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 20s, 53 ℃ of annealing 20s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ of total elongation 10min.
- 7. the fall ill method of former bacterium molecule rapid detection of each described banana bacterial soft rot of claim 1～6 is applied to the quarantine of banana seedling, the early diagnosis of field squirter and monitoring and the evaluation of germ.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789443A (en) * | 2014-02-25 | 2014-05-14 | 广东省农业科学院植物保护研究所 | Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora |
| CN106222106A (en) * | 2016-07-29 | 2016-12-14 | 华南农业大学 | Dick Salmonella and application thereof is proposed until dawn for prevent and treat bacterial wilt |
| CN109182566A (en) * | 2018-09-21 | 2019-01-11 | 广东省农业科学院植物保护研究所 | A method of prevention and treatment banana dasheen bacterial soft rot |
-
2011
- 2011-01-10 CN CN 201110003516 patent/CN102154462B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《bacteriology》 20071231 Bing Ma,Michael等 Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya 第1150－1162页 1－7 第97卷, 第9期 * |
| 《plant disease》 20081231 Wendy S Kaneshiro等 characterization of Erwinia chrysanthemi from a bacterial Heart Rot of Pineapple Outbreak in Hawaii 第92卷, 第10期 * |
| 《plant disease》 20100531 B.R.Lin First report of a soft rot of banana in mainland China caused by a dickeya sp.(Pectobacterium chrysanthemi) 第640页 1－7 第94卷, 第5期 * |
| 《西南林学院学报》 20100630 黄艳霞 菊基腐病菌的常用检测技术 第26－29页 1－7 第30卷, * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789443A (en) * | 2014-02-25 | 2014-05-14 | 广东省农业科学院植物保护研究所 | Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora |
| CN103789443B (en) * | 2014-02-25 | 2015-04-22 | 广东省农业科学院植物保护研究所 | Multiple intermolecular rapid detection method for banana wilt bacteria and bacterial Erwinia carotovora |
| CN106222106A (en) * | 2016-07-29 | 2016-12-14 | 华南农业大学 | Dick Salmonella and application thereof is proposed until dawn for prevent and treat bacterial wilt |
| CN106222106B (en) * | 2016-07-29 | 2019-07-09 | 华南农业大学 | Dick Salmonella and its application are proposed until dawn for prevent and treat bacterial wilt |
| CN109182566A (en) * | 2018-09-21 | 2019-01-11 | 广东省农业科学院植物保护研究所 | A method of prevention and treatment banana dasheen bacterial soft rot |
| CN109182566B (en) * | 2018-09-21 | 2021-07-20 | 广东省农业科学院植物保护研究所 | Method for preventing and treating bacterial soft rot of canna edulis ker |
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