CN106018302A - Application of asparagus lettuce in qualitative and quantitative detection of rhizoctonia toxins - Google Patents
Application of asparagus lettuce in qualitative and quantitative detection of rhizoctonia toxins Download PDFInfo
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- CN106018302A CN106018302A CN201610514691.6A CN201610514691A CN106018302A CN 106018302 A CN106018302 A CN 106018302A CN 201610514691 A CN201610514691 A CN 201610514691A CN 106018302 A CN106018302 A CN 106018302A
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- 239000003053 toxin Substances 0.000 title claims abstract description 74
- 231100000765 toxin Toxicity 0.000 title claims abstract description 74
- 241001361634 Rhizoctonia Species 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 108700012359 toxins Proteins 0.000 title abstract 8
- 244000046738 asparagus lettuce Species 0.000 title abstract 5
- 235000006705 asparagus lettuce Nutrition 0.000 title abstract 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 72
- 239000011574 phosphorus Substances 0.000 claims abstract description 72
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 72
- 238000000034 method Methods 0.000 claims abstract description 33
- 230000003020 moisturizing effect Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 70
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- 206010015866 Extravasation Diseases 0.000 claims description 21
- 230000036251 extravasation Effects 0.000 claims description 21
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 18
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 14
- 229940010552 ammonium molybdate Drugs 0.000 claims description 14
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 14
- 239000011609 ammonium molybdate Substances 0.000 claims description 14
- 238000002835 absorbance Methods 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 11
- 235000010265 sodium sulphite Nutrition 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 238000012545 processing Methods 0.000 claims description 6
- 235000011149 sulphuric acid Nutrition 0.000 claims description 6
- 239000001117 sulphuric acid Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 238000004364 calculation method Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 230000003534 oscillatory effect Effects 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 230000003902 lesion Effects 0.000 claims description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 240000007594 Oryza sativa Species 0.000 description 24
- 235000007164 Oryza sativa Nutrition 0.000 description 23
- 235000009566 rice Nutrition 0.000 description 20
- 241000196324 Embryophyta Species 0.000 description 7
- 206010039509 Scab Diseases 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 101710138657 Neurotoxin Proteins 0.000 description 5
- 239000002581 neurotoxin Substances 0.000 description 5
- 231100000618 neurotoxin Toxicity 0.000 description 5
- UKTDQTGMXUHPIF-UHFFFAOYSA-N [Na].S(O)(O)=O Chemical compound [Na].S(O)(O)=O UKTDQTGMXUHPIF-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- 210000000582 semen Anatomy 0.000 description 4
- 244000061176 Nicotiana tabacum Species 0.000 description 3
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 241001530056 Athelia rolfsii Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of asparagus lettuce in qualitative and quantitative detection of rhizoctonia toxins. Fresh leaves of asparagus lettuce are taken to be subjected to moisturizing cultivation to be used for qualitatively detecting rhizoctonia toxins. Fresh leaves of asparagus lettuce are taken to be used for quantitatively detecting the rhizoctonia toxins by a phosphorus exosmosis method. The asparagus lettuce can be cultivated all the year round, and can be randomly used for determining the activity of toxins, and can be used for quantitatively analyzing the activity of toxins by combining the phosphorus exosmosis method. A method for qualitatively and quantitatively studying the activity of rhizoctonia toxins is convenient and quick, time-saving and labor-saving. A new way is provided for the qualitative and quantitative detection of rhizoctonia toxins.
Description
Technical field
The present invention relates to that rhizoctonia toxin is qualitative and quantitative measurement technology field, particularly relate to a kind of Caulis et Folium Lactucae sativae at rhizoctonia poison
Application in the qualitative and detection by quantitative of element.
Background technology
Toxin, as the important virulence factor of most pathogen, is believed to as selecting pressure replacement pathogen process to post
Main plant tissue or organ, screen the plant of resistance to toxin, it is thus achieved that excellent resistant varieties.At the Oryza sativa L. Resistence research to banded sclerotial blight
In, simplify sharp eyespot resistance authentication method, improve efficiency, and guarantee that result is one of direction of probing into of researcher accurately and reliably.
The resistance of rice sheath blight disease is the most affected by environment, although its field test result is the most directly perceived, but workload is big, is not suitable for disease-resistant
Large-scale operation in breeding.Meanwhile, research show toxin can as identify rice sheath blight disease an important references index,
Can be quantitative with contratoxin, investigation standard is objective, has preferable application prospect in large-scale breeding for disease resistance.About Oryza sativa L. stricture of vagina
Rot verticillium toxin qualitative and detection by quantitative the material of activity is mainly rice tissue, needs paddy growth to late tillering state to Spike development
At the initial stage, the test period is long, wastes time and energy；And limited by the season of growth, seriously constrain the research of toxin.
Caulis et Folium Lactucae sativae, as common edible vegetable, can be cultivated throughout the year, and winter can be with safe overwintering, and Cultivate administration compares water
Rice is easy, can be used for neurotoxin active at any time and measures, and can be a kind of convenient fast in conjunction with phosphorus element extravasation quantitative analysis neurotoxin active
Qualitative and the quantitative method of prompt, time saving and energy saving research rhizoctonia neurotoxin active.
In prior art, qualitative at rhizoctonia toxin about Caulis et Folium Lactucae sativae also little with the research in detection by quantitative, particularly will
Caulis et Folium Lactucae sativae is qualitative for rhizoctonia toxin and quantitative measurement technology is not yet reported.
Summary of the invention
The purpose of the present invention is that provides a kind of Caulis et Folium Lactucae sativae qualitative and fixed at rhizoctonia toxin to solve the problems referred to above
Application in amount detection.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The present invention includes the qualitative detection of rhizoctonia toxin, the detection by quantitative of rhizoctonia toxin, the preparation of solution and standard
The preparation of intermediate liquid；
The qualitative detection of rhizoctonia toxin: take the fresh leaf of Caulis et Folium Lactucae sativae, in putting into diameter 90mm and being covered with the culture dish of filter paper, adds
10mL sterile purified water moisturizing, inoculates 5 μ L rhizoctonia Raw toxin, using blank cultures extract as comparison, is placed in illumination training
Supporting in case, temperature is 28 DEG C, each 12h of light dark, and humidity is more than 90%, often processes and is repeated 3 times, observed and recorded scab after 3 days
Size；
The detection by quantitative of rhizoctonia toxin: employing phosphorus element extravasation method:
(1) phosphorus standard curve: respectively draw phosphorus standard solution (10 μ g/mL) 0.0mL, 1.0mL, 2.0mL, 3.0mL,
In 4.0mL, 5.0mL, 6.0mL to 15mL scale test tube, be then sequentially added into 2mL ammonium molybdate solution, 1mL sodium sulfite solution,
1mL quinol solution, adds distilled water and is settled to 10mL, mixing, stands 30min, measures its absorbance at wavelength 660nm,
Thus calculate regression coefficient, utilize regression equation calculation or be depicted as calibration trace；
(2) measure: taking the centrifuge tube of 5mL, divisional processing and comparison two groups, matched group centrifuge tube adds blank cultures extract
Solution 3mL, process group adds toxin solution 3mL；Take Caulis et Folium Lactucae sativae blade 0.10g to be placed in toxin dilution (extension rate is according to actual feelings
Condition suitably adjusts) in, slight oscillatory so that it is submerge in treatment fluid, child care 24h in 25 DEG C of illumination boxs；Take soak 2mL in
In centrifuge tube, centrifugal 10min under 10000r/min；Take supernatant 0.40mL, be sequentially added into 2mL ammonium molybdate solution, 1mL sulfurous acid
Sodium solution, 1mL quinol solution, add distilled water and be settled to 10mL, mixing, stands 30min, at wavelength 660nm, with corresponding
Toxin soiutions returns to zero, and measures its absorbance, and according to the absorbance measured phosphorus content in sample on standard curve；Often process
It is repeated 5 times；Phosphorus element extravasation rate (%) relatively=(processing phosphorus content-comparison phosphorus content)/comparison phosphorus content × 100；
The preparation of solution:
[1] 15% (V/V) sulfuric acid solution: take 15mL sulphuric acid and be slowly added in 80mL water, and be settled to 100mL；
[2] 5% (W/V) ammonium molybdate solution: take 5g ammonium molybdate, be diluted to 100mL with 15% sulfuric acid solution；
[3] quinol solution: take 0.5g hydroquinone in 100mL water, add a concentrated sulphuric acid after dissolving；
[4] 20% (W/V) sodium sulfite solution (note: this solution need to before every time experiment Extemporaneous): weigh 20g sulfurous
Acid sodium, dissolves with distilled water and is settled to 100mL；
[5] State center for standard matter provides: phosphorus Standard Stock solutions, concentration is 1000 μ g/mL；
The preparation of standard intermediate liquid: the storing solution of phosphorus to be prepared, by potassium dihydrogen phosphate in 105 DEG C of dry 2h, exsiccator
Weigh 0.2195g after interior cooling be dissolved in water and be diluted to 100mL；This storing solution 1mL total phosphorus Han 0.5mg；Pipette storing solution
10.0mL, is diluted with water to 500mL, this solution 1mL total phosphorus Han 0.010mg, uses preparation on the same day；Draw 1mL phosphorus standard inventory
Solution, then moves in 100mL volumetric flask, is settled to 100mL with deionized water, and concentration is 10mg/L.
The beneficial effects of the present invention is:
To be a kind of Caulis et Folium Lactucae sativae qualitative at rhizoctonia toxin and application in detection by quantitative for the present invention, compared with prior art, and this
Invent and Caulis et Folium Lactucae sativae utilization in the qualitative of rhizoctonia toxin and detection by quantitative has been had brand-new understanding, for determining of rhizoctonia toxin
Property and detection by quantitative provide a kind of new experiment material.Caulis et Folium Lactucae sativae can be cultivated throughout the year, can be used for neurotoxin active at any time and measures,
Can be that a kind of convenient and swift, time saving and energy saving research rhizoctonia toxin is lived in conjunction with phosphorus element extravasation method quantitative analysis neurotoxin active
Qualitative and the quantitative method of property.
Accompanying drawing explanation
Fig. 1 is that Raw toxin of the present invention processes different plant leaf blade symptom figure；
In Fig. 1: A: Herba Sonchi Oleracei；B: Plantula Brassicae chinensis；C: Herba Spinaciae；D: Caulis et Folium Lactucae sativae；E: Caulis et Folium Lactucae Sativae；F: Nicotiana tabacum L.
Fig. 2 is phosphorus canonical plotting；
Fig. 3 is that toxin affects comparison diagram to what rice leaf sheath, blade phosphorus element exosmosed；
Fig. 4 is that toxin affects comparison diagram to what Semen Maydis, Caulis et Folium Lactucae sativae phosphorus element exosmosed；
Fig. 5 is that active part inoculates Comparative result figure；
In Fig. 5: A:CK (Richard culture fluid extract)；B: toxin,
Fig. 6 is the correlation curve figure of Caulis et Folium Lactucae sativae and rice leaf sheath phosphorus element volumes of extravasation result.
Detailed description of the invention
The invention will be further described below in conjunction with the accompanying drawings:
The present invention includes the qualitative detection of rhizoctonia toxin, the detection by quantitative of rhizoctonia toxin, the preparation of solution and standard
The preparation of intermediate liquid；
The qualitative detection of rhizoctonia toxin: take the fresh leaf of Caulis et Folium Lactucae sativae, in putting into diameter 90mm and being covered with the culture dish of filter paper, adds
10mL sterile purified water moisturizing, inoculates 5 μ L rhizoctonia Raw toxin, using blank cultures extract as comparison, is placed in illumination training
Supporting in case, temperature is 28 DEG C, each 12h of light dark, and humidity is more than 90%, often processes and is repeated 3 times, observed and recorded scab after 3 days
Size；
The detection by quantitative of rhizoctonia toxin: employing phosphorus element extravasation method:
(1) phosphorus standard curve: respectively draw phosphorus standard solution (10 μ g/mL) 0.0mL, 1.0mL, 2.0mL, 3.0mL,
In 4.0mL, 5.0mL, 6.0mL to 15mL scale test tube, be then sequentially added into 2mL ammonium molybdate solution, 1mL sodium sulfite solution,
1mL quinol solution, adds distilled water and is settled to 10mL, mixing, stands 30min, measures its absorbance at wavelength 660nm,
Thus calculate regression coefficient, utilize regression equation calculation or be depicted as calibration trace；
(2) measure: taking the centrifuge tube of 5mL, divisional processing and comparison two groups, matched group centrifuge tube adds blank cultures extract
Solution 3mL, process group adds toxin solution 3mL；Take Caulis et Folium Lactucae sativae blade 0.10g to be placed in toxin dilution (extension rate is according to actual feelings
Condition suitably adjusts) in, slight oscillatory so that it is submerge in treatment fluid, child care 24h in 25 DEG C of illumination boxs；Take soak 2mL in
In centrifuge tube, centrifugal 10min under 10000r/min；Take supernatant 0.40mL, be sequentially added into 2mL ammonium molybdate solution, 1mL sulfurous acid
Sodium solution, 1mL quinol solution, add distilled water and be settled to 10mL, mixing, stands 30min, at wavelength 660nm, with corresponding
Toxin soiutions returns to zero, and measures its absorbance, and according to the absorbance measured phosphorus content in sample on standard curve；Often process
It is repeated 5 times；Phosphorus element extravasation rate (%) relatively=(processing phosphorus content-comparison phosphorus content)/comparison phosphorus content × 100；
The preparation of solution:
[6] 15% (V/V) sulfuric acid solution: take 15mL sulphuric acid and be slowly added in 80mL water, and be settled to 100mL；
[7] 5% (W/V) ammonium molybdate solution: take 5g ammonium molybdate, be diluted to 100mL with 15% sulfuric acid solution；
[8] quinol solution: take 0.5g hydroquinone in 100mL water, add a concentrated sulphuric acid after dissolving；
[9] 20% (W/V) sodium sulfite solution (note: this solution need to before every time experiment Extemporaneous): weigh 20g sulfurous
Acid sodium, dissolves with distilled water and is settled to 100mL；
[10] State center for standard matter provides: phosphorus Standard Stock solutions, concentration is 1000 μ g/mL；
The preparation of standard intermediate liquid: the storing solution of phosphorus to be prepared, by potassium dihydrogen phosphate in 105 DEG C of dry 2h, exsiccator
Weigh 0.2195g after interior cooling be dissolved in water and be diluted to 100mL；This storing solution 1mL total phosphorus Han 0.5mg；Pipette storing solution
10.0mL, is diluted with water to 500mL, this solution 1mL total phosphorus Han 0.010mg, uses preparation on the same day；Draw 1mL phosphorus standard inventory
Solution, then moves in 100mL volumetric flask, is settled to 100mL with deionized water, and concentration is 10mg/L.
Embodiment 1:
The qualitative detection of rhizoctonia toxin: take the fresh leaf of Caulis et Folium Lactucae sativae, and with Plantula Brassicae chinensis, Herba Sonchi Oleracei, Caulis et Folium Lactucae Sativae, Herba Spinaciae and fresh tobacco leaf be
Comparison, puts in the culture dish being covered with filter paper (diameter 90mm), adds 10mL sterile purified water moisturizing, inoculates 5 μ L rhizoctonias thick
Toxin, using blank cultures extract as comparison, is placed in illumination box, and temperature is 28 DEG C, and each 12h of light dark is wet
Degree, more than 90%, often processes and is repeated 3 times, observed and recorded Lesion size after 3d.
All can with Fig. 1: the AG-1 Raw toxin merging group IA with IB different subgroup rhizoctonia bacterial strain from the data of table 1
Making Herba Sonchi Oleracei, Plantula Brassicae chinensis, Herba Spinaciae, Caulis et Folium Lactucae sativae, Caulis et Folium Lactucae Sativae and Nicotiana tabacum L. excised leaf produce scab, different plant occurring degrees is different；
In vitro Caulis et Folium Lactucae sativae blade contratoxin is most sensitive, and scab is the most obvious, is secondly Caulis et Folium Lactucae Sativae.Pole in Herba Sonchi Oleracei, Plantula Brassicae chinensis and Herba Spinaciae seeded process
Easily occur to rot, affect the evaluation of contratoxin inoculation result；In vitro Caulis et Folium Lactucae sativae blade is qualitative and detection by quantitative as rhizoctonia toxin
Indicator plant, have that rapid onset, sensitivity is high, the most perishable in seeded process, be prone to the advantages such as Cultivate administration, and Oryza sativa L.
Plant or tissue morbidity are in relatively slow, and Rice Cropping management is loaded down with trivial details, higher to season and temperature requirement.In vitro Caulis et Folium Lactucae sativae blade
Can be as rice plant or the substitution material of tissue, as rhizoctonia verticillium toxin qualitative detection is optimal indicator plant.
Table 1 Raw toxin processes different plant leaf scab size
Note: in table, data are meansigma methods ± standard error (SE)；Colleague's lower case difference represents significant difference (P 0.05),
Same column capitalization difference represents significant difference (P 0.05), lower same.
The detection by quantitative of rhizoctonia toxin: use phosphorus element extravasation method.
(1) phosphorus standard curve: respectively draw phosphorus standard solution (10 μ g/mL) 0.0mL, 1.0mL, 2.0mL, 3.0mL,
In 4.0mL, 5.0mL, 6.0mL to 15mL scale test tube, be then sequentially added into 2mL ammonium molybdate solution, 1mL sodium sulfite solution,
1mL quinol solution, adds distilled water and is settled to 10mL, mixing, stands 30min, measures its absorbance at wavelength 660nm,
Thus calculate regression coefficient, utilize regression equation calculation or be depicted as calibration trace.
(2) measure: taking the centrifuge tube of 5mL, divisional processing and comparison two groups, matched group centrifuge tube adds blank cultures extract
Solution 3mL, process group adds toxin solution 3mL.Take Caulis et Folium Lactucae sativae blade 0.10g, and using rice leaf sheath (sheet) and maize leaf as right
According to, it is placed in toxin dilution (diluting 10 times), slight oscillatory so that it is submerge in treatment fluid, child care in 25 DEG C of illumination boxs
24h.Take soak 2mL in centrifuge tube, centrifugal 10min under 10000r/min.Take supernatant 0.30～0.40mL, add successively
Enter 2mL ammonium molybdate solution, 1mL sodium sulfite solution, 1mL quinol solution, add distilled water and be settled to 10mL, mixing, stand
30min, at wavelength 660nm, returns to zero with corresponding toxin soiutions, measures its absorbance, and according to the absorbance measured in standard
Phosphorus content in sample on curve.Often process and be repeated 5 times.Phosphorus element extravasation rate (%) relatively=(process phosphorus content-comparison phosphorus to contain
Amount)/comparison phosphorus content × 100.
Phosphorus element extravasation method is used to determine different strains toxin to rice leaf sheath and the damage ratio of blade cell film, phosphorus standard
Shown in curve chart 2, at the concentration of phosphorus element and 660nm, light absorption value is good linear relationship, and linear equation is y=8.9572x (R2
=0.9972).Rice leaf sheath and blade phosphorus element are exosmosed impact as shown in Table 2 and Figure 3 by different strains toxin.
Table 2 toxin is on rice leaf sheath, the impact of blade phosphorus element extravasation rate
Note: in Fig. 3, data are meansigma methods ± standard error (SE)；Identical bacterial strain lower case difference represents significant difference (P
0.05), different strains capitalization difference represents significant difference (P 0.05), lower same.
Table 2 and Fig. 3 shows, different strains toxin all can make rice leaf sheath and blade phosphorus element exosmose, causing a disease of bacterial strain
Power is the strongest, and phosphorus element extravasation rate is the highest.AG-1IA M-9-11 is the highest to rice leaf sheath and blade phosphorus element extravasation rate；AG-1IB HX-
4C is the most weak to rice leaf sheath and blade phosphorus element extravasation rate.
Table 3 toxin is on Caulis et Folium Lactucae sativae, the impact of maize leaf phosphorus element extravasation rate
Table 3 and Fig. 4 shows, different strains toxin all can make Semen Maydis, Caulis et Folium Lactucae sativae blade phosphorus element exosmose, the pathogenicity of bacterial strain
The strongest, phosphorus element extravasation rate is the highest.AG-1IA M-9-11 is the highest to Semen Maydis, Caulis et Folium Lactucae sativae blade phosphorus element extravasation rate；AG-1IB HX-4C couple
Semen Maydis, Caulis et Folium Lactucae sativae blade phosphorus element extravasation rate are the most weak.
Embodiment 2:
Toxin qualitative detection, rhizoctonia toxin is inoculated in Caulis et Folium Lactucae sativae blade, and blade surface can be made to form obvious scab (Fig. 5).
A:CK (Richard culture fluid extract) in figure；B: toxin.
Embodiment 3:
Toxin qualitative detection, rhizoctonia toxin is inoculated in Caulis et Folium Lactucae sativae blade and rice leaf sheath, outside Caulis et Folium Lactucae sativae is with rice leaf sheath phosphorus element
Milliosmolarity paired samples correlation coefficient and Pearson dependency, as shown in Figure 6.
Toxin processes Caulis et Folium Lactucae sativae blade and rice leaf sheath, and Caulis et Folium Lactucae sativae blade and rice leaf sheath phosphorus element all can be made to exosmose.Caulis et Folium Lactucae sativae
Being notable positive correlation with rice leaf sheath phosphorus element volumes of extravasation result, Pearson correlation coefficient is 0.994, and significance (bilateral) is 0.001,
Linear equation is respectively y=0.9334x (R2=0.9805)；Show Caulis et Folium Lactucae sativae blade can as the substitution material of rice leaf sheath for
Qualitative and the detection by quantitative (Fig. 6) of rhizoctonia toxin.
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described.The skill of the industry
The art personnel simply explanation it should be appreciated that the present invention is not restricted to the described embodiments, described in above-described embodiment and description
The principle of the present invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, these
Changes and improvements both fall within scope of the claimed invention.Claimed scope by appending claims and
Its equivalent defines.
Claims (1)
1. a Caulis et Folium Lactucae sativae is qualitative at rhizoctonia toxin and application in detection by quantitative, it is characterised in that: include rhizoctonia toxin
Qualitative detection, the detection by quantitative of rhizoctonia toxin, the preparation of solution and the preparation of standard intermediate liquid；
The qualitative detection of rhizoctonia toxin: take the fresh leaf of Caulis et Folium Lactucae sativae, in putting into diameter 90mm and being covered with the culture dish of filter paper, adds 10mL
Sterile purified water moisturizing, inoculates 5 μ L rhizoctonia Raw toxin, using blank cultures extract as comparison, is placed in illumination box
In, temperature is 28 DEG C, each 12h of light dark, and humidity is more than 90%, often processes and is repeated 3 times, observed and recorded Lesion size after 3 days；
The detection by quantitative of rhizoctonia toxin: employing phosphorus element extravasation method:
(1) phosphorus standard curve: respectively draw phosphorus standard solution (10 μ g/mL) 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL,
In 5.0mL, 6.0mL to 15mL scale test tube, then it is sequentially added into 2mL ammonium molybdate solution, 1mL sodium sulfite solution, 1mL to benzene
Two phenol solutions, add distilled water and are settled to 10mL, mixing, stand 30min, measure its absorbance, thus calculate at wavelength 660nm
Go out regression coefficient, utilize regression equation calculation or be depicted as calibration trace；
(2) measure: taking the centrifuge tube of 5mL, divisional processing and comparison two groups, matched group centrifuge tube adds blank cultures extract solution
3mL, process group adds toxin solution 3mL；Take Caulis et Folium Lactucae sativae blade 0.10g to be placed in toxin dilution, slight oscillatory so that it is process of submerging
In liquid, child care 24h in 25 DEG C of illumination boxs；Take soak 2mL in centrifuge tube, centrifugal 10min under 10000r/min；Take
Stillness of night 0.40mL, is sequentially added into 2mL ammonium molybdate solution, 1mL sodium sulfite solution, 1mL quinol solution, adds distilled water constant volume
To 10mL, mixing, stand 30min, at wavelength 660nm, return to zero with corresponding toxin soiutions, measure its absorbance, and according to survey
The absorbance gone out is the phosphorus content in sample on standard curve；Often process and be repeated 5 times；Phosphorus element extravasation rate (%) relatively=(process
Phosphorus content-comparison phosphorus content)/comparison phosphorus content × 100；
The preparation of solution:
[1] 15% (V/V) sulfuric acid solution: take 15mL sulphuric acid and be slowly added in 80mL water, and be settled to 100mL；
[2] 5% (W/V) ammonium molybdate solution: take 5g ammonium molybdate, be diluted to 100mL with 15% sulfuric acid solution；
[3] quinol solution: take 0.5g hydroquinone in 100mL water, add a concentrated sulphuric acid after dissolving；
[4] 20% (W/V) sodium sulfite solution: weigh 20g sodium sulfite, dissolve with distilled water and be settled to 100mL；
[5] State center for standard matter provides: phosphorus Standard Stock solutions, concentration is 1000 μ g/mL；
The preparation of standard intermediate liquid: the storing solution of phosphorus to be prepared, by potassium dihydrogen phosphate in 105 DEG C of dry 2h, cold in exsiccator
Weigh 0.2195g the most afterwards be dissolved in water and be diluted to 100mL；This storing solution 1mL total phosphorus Han 0.5mg；Pipette storing solution 10.0mL, use
Water is diluted to 500mL, this solution 1mL total phosphorus Han 0.010mg, uses preparation on the same day；Draw 1mL phosphorus Standard Stock solutions, then
Moving in 100mL volumetric flask, be settled to 100mL with deionized water, concentration is 10mg/L.
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