CN102321620B - Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof - Google Patents
Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof Download PDFInfo
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- CN102321620B CN102321620B CN 201110219702 CN201110219702A CN102321620B CN 102321620 B CN102321620 B CN 102321620B CN 201110219702 CN201110219702 CN 201110219702 CN 201110219702 A CN201110219702 A CN 201110219702A CN 102321620 B CN102321620 B CN 102321620B
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Abstract
The invention discloses a molecular standard sample for bull's eye rot bacteria on apples and a preparation method thereof, and belongs to the field of the research on plant quarantine technologies. The molecular standard sample for the bull's eye rot bacteria on the apples is prepared from high-purity deoxyribonucleic acid (DNA) extracted from bacterial strains of the bull's eye rot bacteria on the apples. The standard sample has the following physical and chemical properties: (1) the standard sample is pure white powder in shape; (2) the content in each tube is 5+/-0.5mu g; (3) the purity of DNA is that OD260/280 is equal to 1.8 to 2; and (4) the standard sample is easily soluble in water or a TE (Tris-EDTA) buffer solution. The molecular standard sample for the bull's eye rot bacteria on the apples is obtained by the following steps of: culturing the bacterial strains, which are taken raw materials, of the bull's eye rot bacteria on the apples in a large quantity under specific conditions, extracting the high-purity DNA, split-charging, and freeze-drying to obtain a molecular standard of the molecular standard sample for the bull's eye rot bacteria on the apples. The standard sample has excellent physical and chemical properties and can be detected by using a specific primer by a polymerase chain reaction (PCR) method, and the sensitivity is 4 to 10 times. Through detection tests, the DNA of the standard sample has high integrity, uniformity and stability; and the standard used as a positive reference substance can be used for import and export detection so as to improve the accuracy of detection results.
Description
Technical field
The present invention relates to apple buphthalmos fruit rot bacterium molecule standard model, also relate in addition its preparation method, belong to Plant Quarantine technical study field.
Background technology
A lot of about the report of animal virus and food microorganisms reference material both at home and abroad, the reference material research of phytopathogen is then at the early-stage, and a lot of problems all need to be resolved hurrily.The bacterial strains of American Type Culture Collecti (ATCC) of buying as positive control more when phytopathogen detected both at home and abroad, its price is very expensive, domesticly almost can't buy, and the rarely seen correlative study report of the molecular dna reference material of phytopathogen Molecular Detection, so comprehensive deep technology of preparing and the stable assurance technology of researching and solving phytopathogen detection DNA standard model, actively develop the development of China Plant Quarantine pathogenic bacteria detection molecules DNA standard model, replenish the blank of this fields of measurement, have very important realistic meaning.
Summary of the invention
The purpose of this invention is to provide apple buphthalmos fruit rot bacterium molecule standard model, the DNA integrity is good, and sample homogeneity, stability are by force; The present invention also provides its preparation method in addition, and technique is simple, and easy handling is mixed with the power height.
Apple buphthalmos fruit rot bacterium molecule standard model of the present invention is characterized in that: make take apple buphthalmos fruit rot bacteria strain extraction high purity DNA as raw material, standard model has following physico-chemical property: (1) shape: pure white is Powdered; (2) the content 5 μ g of every pipe ± 0.5 μ g; (3) DNA purity: OD260/280=1.8～2; (4) soluble in water, or TE(Tris-EDTA) in the damping fluid.
Described preferred DNA purity: OD260/280=1.99.
The preparation method of apple buphthalmos fruit rot bacterium molecule standard model of the present invention comprises the steps:
(1) apple buphthalmos fruit rot bacteria strain adopts potato sucrose (PD) liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days;
(2) extract apple buphthalmos fruit rot bacterium DNA, adopt the CTAB method to extract DNA;
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g ± 0.5 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 100-400 pipe, and then freeze-drying is the purpose standard substance.
Described extraction apple buphthalmos fruit rot bacterium DNA detailed process is:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml (making simultaneously multitube);
2. the CTAB Extraction buffer that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1., the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down gently mixing, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. to 3. adding and the isopyknic chloroform of this supernatant liquor in the middle gained supernatant liquor: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down gently mixing, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. to 4. adding and the isopyknic Virahol of this supernatant liquor or 2 times of volumes ice dehydrated alcohols in the middle gained supernatant liquor, put upside down gently mixing, the DNA flocks appears, be placed on and place 30 min precipitation DNA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm abandons supernatant, and centrifuge tube is inverted on the filter paper dry, with 800 μ l, 70 % ice ethanol washing and precipitating twice, to remove salt ion etc.After adding 800 μ l, 70 % ice ethanol, put upside down gently centrifuge tube several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. to 5. adding 200 μ l TE dissolving DNAs precipitation in the middle gained DNA precipitation.After fully dissolving, add 3 μ l RNase (10 mg/ml), 37 ℃ of water-bath 30 min; Then process by 3.-5. going on foot the same treatment method;
7. with the 6. dry DNA precipitation of step after processing, dissolve with 100 μ l TE ,-20 ℃ save backup.
The preparation method of described apple buphthalmos fruit rot bacterium molecule standard model to the standard model Qualitative Identification, carries out pcr amplification or qualitative analysis is carried out in order-checking by Auele Specific Primer, confirms that prepared nucleic acid is target DNA:
Get the nucleic acid standard model of preparation, add 50 μ lTE, use as template after the dissolving, carry out PCR and order-checking through adopting Auele Specific Primer Phom I and Phom II, carry out qualitative analysis, confirm that prepared nucleic acid standard model is the purpose nucleic acid samples;
Forward primer: 5'-CTTTCTCCGTTGTCCCATCC-3'
Reverse primer: 5'-GAACATTGCGCATCTGGTCC-3'
Reaction system:
Forward primer 1 μ l
Reverse primer 1 μ l
Taq enzyme 0.2U
dNTP 4 μl
10×Buffer 3 μl
Total 30 μl
Reaction conditions:
94℃ 2min
98℃ 10s
57℃ 30s
72 ℃ of 30s circulate 35 times
72℃ 10min。
Apple buphthalmos fruit rot bacterium molecule standard model of the present invention is take apple buphthalmos fruit rot bacteria strain as raw material, cultivate in a large number by specified conditions, extract high purity DNA, carry out packing, freeze-drying, obtain the molecular criteria product of apple buphthalmos fruit rot bacterium, physico-chemical property is good, can adopt specific primer to detect by PCR method, and sensitivity reaches 10
-4Doubly.After testing test, these standard substance DNA integrity, homogeneity, good stability can be used for importing and exporting in the detection, as positive reference substance, improve the accuracy of detected result.
Four, description of drawings
Fig. 1 is the total DNA electrophorogram of apple buphthalmos fruit rot bacterium.
Fig. 2 is apple buphthalmos fruit rot bacterium primer amplified product electrophoresis result figure, and wherein 1,2,3 is target DNA, and 4 is blank.
Fig. 3 is standard model sensitivity detected result figure of the present invention, and wherein by left-to-right, per two lattice are one group, are 10 successively
-1, 10
-2, 10
-3, 10
-4, 10
-5
Five, embodiment
Below in conjunction with specific embodiment the present invention is described in further detail, but the present invention is not limited to specific embodiment.
Apple buphthalmos fruit rot bacterium molecule standard model and preparation method thereof carries out as follows:
(1) apple buphthalmos fruit rot bacteria strain adopts potato sucrose (PD) liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days.
(2) extract apple buphthalmos fruit rot bacterium DNA, adopt the CTAB method to extract DNA, concrete grammar is:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml (making simultaneously multitube);
2. the CTAB Extraction buffer that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1., the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down gently mixing, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. to 3. adding and the isopyknic chloroform of this supernatant liquor in the middle gained supernatant liquor: primary isoamyl alcohol mixed solution (volume ratio is 24:1), put upside down gently mixing, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. to 4. adding and the isopyknic Virahol of this supernatant liquor or 2 times of volumes ice dehydrated alcohols in the middle gained supernatant liquor, put upside down gently mixing, the DNA flocks appears, be placed on and place 30 min precipitation DNA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm abandons supernatant, and centrifuge tube is inverted on the filter paper dry, with 800 μ l, 70 % ice ethanol washing and precipitating twice, to remove salt ion etc.After adding 800 μ l, 70 % ice ethanol, put upside down gently centrifuge tube several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. to 5. adding 200 μ l TE dissolving DNAs precipitation in the middle gained DNA precipitation.After fully dissolving, add 3 μ l RNase (10 mg/ml), 37 ℃ of water-bath 30 min; Then process by 3.-5. going on foot the same treatment method;
7. with the 6. dry DNA precipitation of step after processing, dissolve with 100 μ l TE ,-20 ℃ save backup.
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g/ pipes, every kind of nucleic acid molecule standard specimen packing 150 pipes, and then freeze-drying is the purpose standard substance, and have following physico-chemical property: shape: pure white is Powdered; The content 5 μ g of every pipe ± 0.5 μ g; DNA purity: OD260/280=1.8～2; Soluble in water, or TE(Tris-EDTA) in the damping fluid.
(4) get the nucleic acid standard model of preparation, add 50 μ lTE, use as template after the dissolving, carry out PCR and order-checking through adopting specificity, carry out qualitative analysis, confirm that prepared nucleic acid standard model is the purpose nucleic acid samples.Example:
Forward primer: 5'-CTTTCTCCGTTGTCCCATCC-3'
Reverse primer: 5'-GAACATTGCGCATCTGGTCC-3'
Reaction system:
Taq enzyme 0.2U
dNTP 4 μl
10×Buffer 3 μl
Total 30 μl
Reaction conditions:
94℃ 2min； 98℃ 10s，57℃ 30s，72℃ 30s，35cycles；72℃ 10min。
The standard model that embodiment 1 is made carries out DNA integrity, homogeneity, stability test:
1.DNA quality and integrity detection
Get the DNA of preparation, carry out gel electrophoresis, purity and concentration and detect.
(1) DNA integrity detection: the direct electrophoretic examinations of DNA that direct method-usefulness is extracted, 120V, 1.5% agarose gel electrophoresis, the integrity of observing band, detected result is seen Fig. 1, band is complete, without the disperse shape, illustrates that DNA band integrity is better.
(2) DNA concentration and purity detecting: ultraviolet spectrophotometer method-absorption 1 μ l DNA is with the absorption value of spectrophotometric determination 260nm and 280nm, then according to OD
260/ OD
280Value is judged DNA purity, and according to OD
260Calculate its concentration.Calculate as follows:
DNA concentration (ng/ μ l)=OD
260* 50
Work as OD
260/ OD
280＜1.8, the expression protein content is higher; OD
260/ OD
280＞2.0, the expression rna content is higher; Work as OD
260/ OD
280=1.8～2.0, DNA is purer in expression.
2. standard model analysis of Uniformity
For checking the homogeneity of the purpose nucleic acid standard model for preparing, adopt PicoGreen dna molecular fluorescent quantitation method and ultraviolet spectrophotometry, get and randomly draw 15 pipe samples, every pipe sample is divided into 2 one's share of expenses for a joint undertaking samples to be tested, and the results are shown in (table 1) data and carries out F check (table 2) confirmatory sample homogeneity.
3. standard model stability analysis
Originally there is the card standard model to have the stability period limit gauge accepted argument of card standard model bright (such as the production regulation explanation of U.S. SIGMA company with reference to external on an equal basis band card, this has the card standard model vacuum, lucifuge ,-20 ℃ of lower storages, stable validity period is 2 years) be foundation, biological nature according to microorganism, scheduled to last with 2 years, standard model to preparation is got two duplicate samples stage by stage at every turn, carries out qualitative test according to different preservation conditions, and every duplicate samples is to measure 3 sub-values as its definite value result.In whole stability tests, used personnel, instrument, testing method and laboratory are all identical with uniformity test.
According to said determination method difference calculation result, this apple buphthalmos fruit rot bacterium standard model is vacuum, lucifuge ,-20 ℃ of lower storages, and stable validity period is 2 years, and measurement result sees Table 3.
In the stability test, slope can calculate with following formula:
Intercept is calculated by following formula:
The standard deviation of the point on the straight line can be calculated by following formula:
Get its square root s=0.09820%, the uncertainty relevant with slope calculated with following formula:
Degree of freedom is n-2=5 and p=0.95(95% confidence level) the student distribution t-factor equal 2.57.
Because
So slope is inapparent.Thereby do not observe unstable in the stability experiment.
Table 1 apple buphthalmos fruit rot bacterium molecule standard substance homogeneity detected result
| | Increment | 1 test result (μ g) | |
Mean value | Standard deviation |
| 1-1 | 5.081 | 5.143 | 5.112 | 0.04384 | |
| 1-2 | 5.0705 | 5.939 | 5.505 | 0.61412 | |
| 1-3 | 5.1475 | 5.1175 | 5.133 | 0.02121 | |
| 1-4 | 5.108 | 5.0855 | 5.097 | 0.01591 | |
| 1-5 | 5.236 | 5.2385 | 5.237 | 0.00177 | |
| 1-6 | 5.1855 | 5.1575 | 5.172 | 0.01980 | |
| 1-7 | 5.3205 | 5.3515 | 5.336 | 0.02192 | |
| 1-8 | 5.331 | 5.3 | 5.316 | 0.02192 | |
| 1-9 | 5.36 | 5.394 | 5.377 | 0.02404 | |
| 1-10 | 4.9895 | 4.9355 | 4.963 | 0.03818 | |
| 1-11 | 5.4775 | 5.4975 | 5.488 | 0.01414 | |
| 1-12 | 5.064 | 5.0695 | 5.067 | 0.00389 | |
| 1-13 | 5.3065 | 5.2795 | 5.293 | 0.01909 | |
| 1-14 | 5.358 | 5.3765 | 5.367 | 0.01308 | |
| 1-15 | 5.112 | 5.113 | 5.113 | 0.00071 |
Table 2 apple buphthalmos fruit rot bacterium molecule standard substance homogeneity F assay
Table 3 apple buphthalmos fruit rot bacterium molecule standard substance Detection of Stability result
SEQUENCE LISTING
＜110〉Liu, slowly
Cao, the border is beautiful
The king, blissful
Lee, prosperous
＜120〉apple buphthalmos fruit rot bacterium molecule standard model and preparation method thereof
<130> 2011102197025
<140> 2011102197025
<141> 2011-08-02
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
＜213〉synthetic
<220>
<221> misc_feature
<222> (1)..(20)
＜223〉forward primer
<400> 1
ctttctccgt tgtcccatcc 20
<210> 2
<211> 20
<212> DNA
＜213〉synthetic
<220>
<221> misc_feature
<222> (1)..(20)
＜223〉reverse primer
<400> 2
gaacattgcg catctggtcc 20
Claims (3)
1. the preparation method of apple buphthalmos fruit rot bacterium molecule standard model is characterized in that: extract high purity DNA as raw material makes take apple buphthalmos fruit rot bacteria strain, comprise the steps:
(1) apple buphthalmos fruit rot bacteria strain adopts the potato sucrose liquid nutrient medium, places under 20～22 ℃ of conditions and cultivates 5～7 days;
(2) extract apple buphthalmos fruit rot bacterium DNA, adopt the CTAB method to extract DNA, comprise the steps:
1. get the dried mycelia of 0.1 g and place the mortar of precooling, in liquid nitrogen, be ground into powder rapidly, powder is changed over to rapidly in the centrifuge tube of 1.5 ml;
2. the CTAB Extraction buffer that adds 65 ℃ of preheatings of 800 μ l in the centrifuge tube of dried mycelia powder is housed in 1., the vortex abundant mixing that vibrates, 65 ℃ of water-bath 50 min put upside down mixing once gently every 10 min;
3. be cooled to room temperature, centrifuge tube adds the chloroform of 800 μ l in 2. again: the primary isoamyl alcohol mixed solution, put upside down gently mixing, and leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
4. to 3. adding and the isopyknic chloroform of this supernatant liquor in the middle gained supernatant liquor: the primary isoamyl alcohol mixed solution, put upside down gently mixing, leave standstill 10 min, 4 ℃ of 10000 centrifugal 10 min of rpm gets supernatant;
5. to 4. adding and the isopyknic Virahol of this supernatant liquor or 2 times of volumes ice dehydrated alcohols in the middle gained supernatant liquor, put upside down gently mixing, the DNA flocks appears, be placed on and place 30 min precipitation DNA on ice, 4 ℃ of 12000 centrifugal 10 min of rpm, abandon supernatant, centrifuge tube is inverted on the filter paper dry, with 800 μ l concentration, 70 % ice ethanol washing and precipitating twice, to remove salt ion: after adding 800 μ l concentration, 70 % ice ethanol, put upside down gently centrifuge tube several times, the DNA that is deposited in the bottom is upspring, place centrifuge tube 10 min, 4 ℃ of 10000 centrifugal 1 min of rpm abandons supernatant; Wash the DNA precipitation with 800 μ l concentration, 70 % ice ethanol again, method is the same, and 4 ℃ of 15000 centrifugal 3 min of rpm abandons supernatant, air-dry removal ethanol;
6. to 5. adding 200 μ l TE dissolving DNAs precipitation in the middle gained DNA precipitation, after fully dissolving, add the RNase of 3 μ l concentration, 10 mg/ml, 37 ℃ of water-bath 30 min; Then process by 3.-5. going on foot the same treatment method;
7. with the 6. dry DNA precipitation of step after processing, dissolve with 100 μ l TE ,-20 ℃ save backup;
(3) preparation of nucleic acid standard model: the DNA that will measure after the concentration carries out packing by 5 μ g ± 0.5 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 100-400 pipe, then freeze-drying is the purpose standard substance, and standard model has following physico-chemical property: (1) shape: pure white is Powdered; (2) the content 5 μ g of every pipe ± 0.5 μ g; (3) DNA purity: OD260/280=1.8～2; (4) soluble in water, or TE(Tris-EDTA) in the damping fluid.
2. the preparation method of apple buphthalmos fruit rot bacterium molecule standard model according to claim 1, it is characterized in that: to the standard model Qualitative Identification, carry out pcr amplification or qualitative analysis is carried out in order-checking by Auele Specific Primer, confirm that prepared nucleic acid is target DNA:
Get the nucleic acid standard model of preparation, add 50 μ lTE, use as template after the dissolving, carry out PCR and order-checking through adopting Auele Specific Primer Phom I and Phom II, carry out qualitative analysis, confirm that prepared nucleic acid standard model is the purpose nucleic acid samples;
Forward primer: 5'-CTTTCTCCGTTGTCCCATCC-3'
Reverse primer: 5'-GAACATTGCGCATCTGGTCC-3'
Reaction system:
Template 1～2 μ l
Forward primer 1 μ l
Reverse primer 1 μ l
Taq enzyme 0.2U
dNTP 4 μl
10×Buffer 3 μl
Total 30 μl
Reaction conditions:
94℃ 2min
98℃ 10s
57℃ 30s
72 ℃ of 30s circulate 35 times
72℃ 10min。
3. the preparation method of apple buphthalmos fruit rot bacterium molecule standard model according to claim 1 is characterized in that: the chloroform that adopts among the described extraction apple buphthalmos fruit rot bacterium DNA: the primary isoamyl alcohol mixeding liquid volume is than being 24:1.
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|---|---|---|---|
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| CN104630376A (en) * | 2015-02-26 | 2015-05-20 | 李鑫 | LAMP primer group for detecting pathogenic bacteria of bull's eye rot on apple and application thereof |
| CN112710523B (en) * | 2020-12-28 | 2023-09-19 | 山西省农业科学院农作物品种资源研究所 | Collecting device suitable for molecular marker detects |
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| CN101037704A (en) * | 2006-12-30 | 2007-09-19 | 福建省农业科学院生物技术研究所 | Detecting method for fusarium oxysporum pathogenicless strain |
| CN101182344A (en) * | 2007-12-10 | 2008-05-21 | 贵州省果树科学研究所 | Method for extracting and purifying DNA of plants like cactus |
| CN101603082A (en) * | 2009-04-17 | 2009-12-16 | 广州甘蔗糖业研究所 | The PCR method for quick of sugarcane ratoon stunting disease bacterium |
| CN101701252A (en) * | 2009-11-20 | 2010-05-05 | 吉林大学 | Kit for rapidly detecting staphylococcus aureus in milk |
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2011
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037704A (en) * | 2006-12-30 | 2007-09-19 | 福建省农业科学院生物技术研究所 | Detecting method for fusarium oxysporum pathogenicless strain |
| CN101182344A (en) * | 2007-12-10 | 2008-05-21 | 贵州省果树科学研究所 | Method for extracting and purifying DNA of plants like cactus |
| CN101603082A (en) * | 2009-04-17 | 2009-12-16 | 广州甘蔗糖业研究所 | The PCR method for quick of sugarcane ratoon stunting disease bacterium |
| CN101701252A (en) * | 2009-11-20 | 2010-05-05 | 吉林大学 | Kit for rapidly detecting staphylococcus aureus in milk |
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| Title |
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| acute * |
| G. Palm.Resistance of storage rot fungi Neofabraea perennans, N. alba, Glomerella acutata and Neonectria galligena against thiophanate-methyl in Northern German apple production.《Journal of Plant Diseases and Protection》.2010,第117卷(第4期),第186页. * |
| PY.Species specific identification of the Neofabraea pathogen complex associated with pome fruits using PCR and multiplex DNA amplification.《Mycol. Res.》.2003,第107卷(第5期),528–536. * |
| R.W.S. Weber & G. Palm.Resistance of storage rot fungi Neofabraea perennans, N. alba, Glomerella acutata and Neonectria galligena against thiophanate-methyl in Northern German apple production.《Journal of Plant Diseases and Protection》.2010,第117卷(第4期),第186页. |
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