CN107860754A - Soybean browning SCN cyst automatic counting method - Google Patents
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06M—COUNTING MECHANISMS; COUNTING OF OBJECTS NOT OTHERWISE PROVIDED FOR
- G06M11/00—Counting of objects distributed at random, e.g. on a surface
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Abstract
The invention discloses a kind of soybean browning SCN cyst automatic counting method.The present invention provides a kind of nematode cyst method of counting, comprises the following steps：Counted after being taken pictures using single excitation source to testing sample.This method make use of the principle of cyst auto-fluorescence imaging, all-wave length halogen light source and xenon light source are instead of using the LASER Light Source of single wavelength, improve the exciting light energy of the excitation wavelength, make under old terms can not the brown cyst of strong luminescence light, the picture of high discrimination is obtained, automatic count is realized in conjunction with image analysis software.The purpose quick and precisely counted can be reached using the method for the present invention, artificial disturbance is excluded, stale sample can be handled, the window phase of cyst counting is extended, be separated into possibility with laboratory with making inoculation.The present invention greatly facilitates the development of SCN research work, has very big application value.
Description
Technical field
The present invention relates to a kind of soybean browning SCN cyst automatic counting method.
Background technology
Soybean is the important sources of people's daily ingestion vegetable protein and vegetable oil, is the important work of the economic people's livelihood of relation
Thing.Soybean cyst nematode Heterodera glycines (Heteroderaglycines Ichinohe, SCN) are that maximum disease is endangered in Soybean production, one
As can cause 5% -10% production loss, seriously band even can be with the underproduction to more than 30%, or even No kernels or seeds are gathered, as in a year of scarcity.Greatly
Beans SCN has the characteristics that into live time length, host range is wide, route of transmission is more, is difficult to prevent and treat in production.People are with female
Sex index (Female Index, FI) is index, the calculation formula of female adult index：
Cyst number × 100 on cyst number/perceptual check variety root on female adult index (FI)=differential variety root.Cause
This, the either excavation of resistant gene, the screening of resistant variety, the research of resistance mechanism all must be with SCN counting skill
Based on art.
Traditional soybean cyst nematode Heterodera glycines method of counting is to treat plant inoculating soybean cyst nematode Heterodera glycines 30 days or so, is contained in aobvious capsule
During the phase, soybean root is dug out completely, rinsed, rubbed to water with flowing water, stirring suspension, sieving, cyst is gone out by screen filtration,
Observe and count under the microscope.This method, which takes, uninteresting is not suitable for batch operation.
2005 Nian You researchers have invented a kind of cyst method of counting based on fluoroscopic imaging systems, and the method can make
Cyst counts highly efficient.The fluoroscopic imaging systems are made up of the photosystem of a closing, a video camera and a computer,
Using halogen light as excitation source.This system needs two wave filters, and one is arranged on light exciting pathway, makes the light of proper strength
Source excitation sample, another is arranged on before zoom lens, the light of suitable wavelength is passed through increase cyst and root etc. impurity letter
Number contrast, so as to obtain high-contrast, clear photograph, recycle photo to carry out artificial counting.This method by with
The cyst of the soybean root system grown in culture medium is inoculated in counting.
Brown in 2010 et al. improves this method, has used xenon light source, and is equipped with the refrigeration CCD of 4,000,000 pixels,
And Image Analysis software.System after improvement can count automatically, the soybean cyst nematode Heterodera glycines that be inoculated in greenhouse can be carried out high
The counting of flux.But this method is mainly used in counting also taint-free fresh cyst.Storage or storage condition are bad for a long time
Good (such as temperature height), can make cyst browning, and fluorescence signal dies down after cyst browning, just be difficult to obtain clearly image knot
Fruit, this defect limit the use of the technology.
The content of the invention
It is an object of the invention to provide a kind of soybean browning SCN cyst automatic counting method.
The invention provides a kind of nematode cyst method of counting, comprise the following steps：Using the excitation source of single wavelength
Counted after being taken pictures to testing sample.
Methods described specifically comprises the following steps：Using the excitation source of single wavelength, testing sample is excited, is made therein
Nematode sporangiocyst lights, and is then taken pictures and is counted.
In methods described, the excitation source of the single wavelength is the excitation source of 470nm or 532nm excitation wavelengths.
In methods described, parameter setting when taking pictures is as follows：
(1) excitation source：470nm or 532nm excitation wavelengths；
(2) filter：535nm, 580nm, 620nm, 660nm or 730nm launch wavelength；
(3) time for exposure：10ms-30s.
Parameter setting when taking pictures is specific as follows：
(1) excitation source：470nm excitation wavelengths；
(2) filter：535nm launch wavelengths；
(3) time for exposure：6s.
Parameter setting when taking pictures is specific as follows：
(1) excitation source：532nm excitation wavelengths；
(2) filter：620nm launch wavelengths；
(3) time for exposure：15s.
Laser output power when taking pictures can be 10%-100%.
Laser output power when taking pictures concretely 80%.
Parameter setting when taking pictures is concretely：Exciting power：5W；Excitation wavelength：532nm；Launch light filter：
620nm；Laser output power：4 tunnels are 80%；Time for exposure：15s.
Parameter setting when taking pictures is concretely：Exciting power：5W；Excitation wavelength：470nm；Launch light filter：
535nm；Laser output power：4 tunnels are 80%；Time for exposure：6s.
Specifically usable light of being taken pictures described in any of the above reflect U.S. board In-vivo Master in body fluoroscopic imaging systems and
Micro-Manager softwares (https://www.micro-manager.org/) realize.
In methods described, the parameter setting during counting concretely area (area) 30-100 pixels；roundness
(circularity) 1-1.2；Size (length) (length) 6-12 pixels；Size (width) (width) 4.8-10 pixels.
The counting can specifically use the softwares of Image-Pro Plus 6.0 to realize.
In methods described, the preparation method of the testing sample comprises the following steps：Originally, water flows through bushing screen (bushing screen for sampling
Upper sieve be 20 mesh, lower sieve is 60 mesh), take the sporangiocyst stayed on 60 mesh sieves to be counted.
The sample concretely plant tissue.It is described to organize concretely root tissue.
The concretely browning nematode packing of nematode packing described in any of the above.
The present invention also protects any of the above methods described in plant identification to the application in nematode resistance.
The present invention also protects a kind of plant identification to comprise the following steps the method for nematode resistance：
(1) by nematode inoculated plant, cultivated；
(2) after completing step (1), taking plant tissue, processing sample obtains sample to be tested, using any of the above as sample
Methods described counts to the nematode in plant tissue；
Resistance of the plant to nematode is judged according to the result of step (2).
The plant tissue concretely plant root tissue.
The concretely cyst roundworm of nematode described in any of the above.The cyst roundworm concretely soy bean cyst roundworm.
Plant described in any of the above can be soybean.
Fluorescence imaging method of the prior art can not count to brown cyst, just can only be according to once cyst changes colour
Manually count, and brown cyst is similar to root impurity color, by being visually difficult to differentiate between, counting just needs under the microscope
Carry out, extremely take time and effort.Count results rely on the proficiency of technical staff very much simultaneously, and human factor influences very big.Secondly people
Work, which counts, is not suitable for mass disposal sample, if processing makes counting inaccurate not in time, virtually limits resistant variety
The scale of screening.Long-distance transport can not keep the fresh of sample, also obstruct researcher to other initiative and advantages of the localities nematode microspecies
Identification research.
This method make use of the principle of cyst auto-fluorescence imaging, and all-wave length is instead of using the LASER Light Source of single wavelength
Halogen light source and xenon light source, the exciting light energy of the excitation wavelength is improved, making under old terms can not strong luminescence
Brown cyst is lighted, and obtains the picture of high discrimination, and automatic count is realized in conjunction with image analysis software.Use the side of the present invention
Method can reach the purpose quick and precisely counted, exclude artificial disturbance, can handle stale sample, extend cyst counting
Window phase, it is separated into possibility with laboratory with making inoculation.The present invention greatly facilitates the development of SCN research work,
With very big application value.
Brief description of the drawings
Fig. 1 is that 470nm excitation wavelengths coordinate different transmitting light filters to take pictures result.
Fig. 2 is that 532nm excitation wavelengths coordinate different transmitting light filters to take pictures result.
Fig. 3 is that 532nm excitation wavelengths coordinate 620nm launch wavelength filters, and different exposure time is taken pictures result.
Fig. 4 is that 470nm excitation wavelengths coordinate 535nm launch wavelength filters, and different exposure time is taken pictures result.
Fig. 5 is that 532nm excitation wavelengths coordinate 620nm launch wavelength filters to take pictures, and different laser output powers are taken pictures result.
Fig. 6 is that embodiment 3 is taken pictures result.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
The susceptible kind Magellan of soybean：Bibliography：The cyst roundworm Resistence research of the good soybean of Liu Shiming, Peng De is new
Be in progress [J] Chinese sciences:Life science, 2016,46 (5):535.；The public can grind from Chinese Academy of Agricultural Sciences oil crops
Study carefully and obtained.
Soy bean cyst roundworm：Bibliography：Chen Gui save, Yan Qingshang, Yan Shurong, wait soy bean cyst roundworms harm and its
Prevent and treat [J] journal of crops, 2000 (1):6-9.；The public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture.
Embodiment 1, soybean browning SCN cyst automatic counting method
First, preparation of samples
Testing sample is taken, root is rinsed with flowing water, water flows through bushing screen (the upper sieve of bushing screen is 20 mesh, and lower sieve is 60 mesh), will
The cyst stayed on 60 mesh sieves is carefully eluted in beaker with wash bottle, is poured into cyst in plate during counting, and it is uniformly divided
Dissipate, plate is then placed on sample stage center.
2nd, take pictures
U.S. board In-vivo Master are reflected using light to take pictures in body fluoroscopic imaging systems.
Exciting power：5W；Excitation wavelength：470nm or 532nm；Launch light filter：535nm、580nm、620nm、660nm
Or 730nm.
(1) 4 road laser output powers, scope 10%-100% are set.
(2) Micro-Manager softwares (https is used://www.micro-manager.org/) carry out observation and take pictures.
Time for exposure is 10ms-30s.
(3) picture is preserved using ImageJ analysis softwares.
3rd, SCN counts
The picture shot with the software opening steps three of Image-Pro Plus 6.0 is analyzed, and method is as follows：
" count and measure objects (count and measure) " instrument is used, is selected in the dialog box of ejection
" manual (manual) ", then " select ranges... (selection range) " is clicked on, new dialog box is ejected, selects red-label
Scope, general right side red mark line acquiescence cause all cysts in picture in low order end, appropriate mobile left side red mark line
All upper red of mark, are then shut off.Original dialog box is now returned to, " measure (measurement) " option is found out in menu bar,
Eject new dialog box, arrange parameter.SCN is generally oval, selects following 4 parameters, " area (faces
Product) ", " roundness (circularity) ", " size (length) (length) ", " size (width) (width) ", the parameter after optimization
It is as follows：Area (area) 30-100 pixels；Roundness (circularity) 1-1.2；Size (length) (length) 6-12 pixels；
Size (width) (width) 4.8-10 pixels.
After setting up parameter, " ok " is clicked in the lower right corner, returns to last dialog box, is clicked on " count (counting) ", you can learn
The quantity of cyst in selected scope.The numerical value for recording In Range in Count/Size dialog boxes is count results.
Embodiment 2, parameter optimization of taking pictures
Testing sample：Cyst on susceptible kind Magellan is grown in 6 months in northeast SCN sick nursery, sample is through field
After sampling 4 degree refrigeration 40 days after (cyst browning) observed.
First, excitation wavelength and transmitting light filter Combinatorial Optimization
1st, SCN cyst is counted using the method for embodiment 1.Parameter setting of taking pictures is as follows：Exciting power：
5W；Excitation wavelength：470nm；Launch light filter：Set three groups (510nm, 535nm and 580nm)；Laser output power：4 roads are equal
For 80%；Time for exposure：3s.
As a result it is as shown in Figure 1.As a result show, 470nm excitation wavelengths coordinate 535nm launch wavelength filters to take pictures effect most
It is good.
2nd, SCN cyst is counted using the method for embodiment 1.Parameter setting of taking pictures is as follows：Exciting power：
5W；Excitation wavelength：532nm；Launch light filter：Set four groups (580nm, 620nm, 660nm and 730nm)；Laser output power：
4 tunnels are 80%；Time for exposure：15s.
As a result it is as shown in Figure 2.As a result show, 532nm excitation wavelengths coordinate 620nm launch wavelength filters to take pictures effect most
It is good.
Summary result, obtain two kinds of effect optimum combinations of taking pictures：(1) 470nm excitation wavelengths coordinate 535nm transmitted waves
Long filter；(2) 532nm excitation wavelengths coordinate 620nm launch wavelength filters.
2nd, the time for exposure optimizes
1st, SCN cyst is counted using the method for embodiment 1.Parameter setting of taking pictures is as follows：Exciting power：
5W；Excitation wavelength：532nm；Launch light filter：620nm；Laser output power：4 tunnels are 80%；Five groups of time for exposure are set：
3s, 6s, 10s, 15s and 25s.
As a result it is as shown in Figure 3.As a result show, when 532nm excitation wavelengths cooperation 620nm launch wavelength filters are taken pictures, exposure
Time 15s best results.
2nd, SCN cyst is counted using the method for embodiment 1.Parameter setting of taking pictures is as follows：Exciting power：
5W；Excitation wavelength：470nm；Launch light filter：535nm；Laser output power：4 tunnels are 80%；Four groups of time for exposure are set：
1s, 2s, 6s and 8s.
As a result it is as shown in Figure 4.As a result show, when 470nm excitation wavelengths cooperation 535nm launch wavelength filters are taken pictures, exposure
Time 6s best results.
3rd, laser output power optimizes
SCN cyst is counted using the method for embodiment 1.Parameter setting of taking pictures is as follows：Exciting power：5W；
Excitation wavelength：532nm；Launch light filter：620nm；Four groups of laser output powers are set：4 tunnels be 20%, 4 tunnels be 40%,
4 tunnels be 80% and 4 tunnel be 100%；Time for exposure：15s.
As a result it is as shown in Figure 5.As a result show, when 532nm excitation wavelengths cooperation 620nm launch wavelength filters are taken pictures, laser
It is 80% best results that power output, which is arranged to 4 tunnels,.
4th, optimal parameter of taking pictures
The optimum results of combining step one, step 2 and step 3, obtain following two groups optimal parameters of taking pictures：
(1) exciting power：5W；Excitation wavelength：532nm；Launch light filter：620nm；Laser output power：4 tunnels are
80%；Time for exposure：15s.
(2) exciting power：5W；Excitation wavelength：470nm；Launch light filter：535nm；Laser output power：4 tunnels are
80%；Time for exposure：6s.
Embodiment 3, actual sample count
Testing sample：Cyst on susceptible kind Magellan is grown in 6 months in northeast SCN sick nursery, sample is through field
After sampling 4 degree refrigeration 40 days after (cyst browning) observed.
First, preparation of samples
According to carrying out sample preparation the step of step 1 in embodiment 1.
2nd, take pictures and count
1st, U.S. board In-vivo Master are reflected using light to take pictures in body fluoroscopic imaging systems.
Exciting power：5W；
Excitation wavelength and transmitting light filter：532nm excitation wavelengths coordinate 620nm launch wavelength filters.
(1) 4 road laser output powers are set, and 4 tunnels are 80%.
(2) Micro-Manager softwares (https is used://www.micro-manager.org/) carry out observation and take pictures.
Time for exposure is 10s.
(3) picture is preserved in ImageJ analysis softwares.
2nd, using Bruker In-Vivo Xtreme system reference literatures：Brown S,Yeckel G,Heinz R,
Clark K,Sleper D,Mitchum MG(2010)A high-throughput automated technique for
counting females of Heteroderaglycines using a fluorescence-based imaging
System.J Nematol42, the method in 201-206. are taken pictures, time for exposure 10s.
As a result it is as shown in Figure 6.Fig. 6 A are the picture of taking pictures that step 1 obtains (arrow meaning is cyst).Fig. 6 B positions step 2
To picture of taking pictures (arrow meaning is cyst).
As a result show, the prior art in document, fluorescence signal dies down after cyst browning, is just difficult to clearly be schemed
Picture result, the picture of higher discrimination can be obtained using the method for the present invention.
3rd, the software that artificial counting and the step 3 of embodiment 1 is respectively adopted counts two methods to 15 groups of steps 1 automatically
Result of taking pictures is counted, the record count time, as a result as shown in Table 1 and Table 2.As a result show, artificial counting is automatic with software
The correlation coefficient r of counting2=0.99.Software count results are accurate and the used time is greatly reduced.
The count results of table 1 count
The gate time of table 2 counts
| Numbering | The artificial used time | Software counts |
| 1 | 1’42” | 18” |
| 2 | 2’10” | 21” |
| 3 | 2’06” | 18” |
| 4 | 2’34” | 21” |
| 5 | 3’06” | 21” |
| 6 | 1’45” | 21” |
| 7 | 2’10” | 21” |
| 8 | 1’22” | 21” |
| 9 | 1’40” | 18” |
| 10 | 2’07” | 21” |
| 11 | 3’44” | 18” |
| 12 | 2’13” | 21” |
| 13 | 2’52” | 18” |
| 14 | 2’24” | 21” |
| 15 | 3’44” | 15” |
| It is average | 2’19” | 19.6” |
Embodiment 4,
The susceptible kind Magellan of soybean is inoculated in soy bean cyst roundworm, collects cyst, (the born of the same parents after 4 degree refrigerate 40 days
Capsule browning), counting of taking pictures is carried out according to the step of embodiment 3, can accurately complete to count, the method can also be used for plant
The Resistance Identification of cyst roundworm and other cyst roundworms are studied.
Claims (10)
1. a kind of nematode cyst method of counting, comprises the following steps：Testing sample is carried out using the excitation source of single wavelength
Counted after taking pictures.
2. the method as described in claim 1, excitation source the swashing for 470nm or 532nm excitation wavelengths of the single wavelength
Light emitting source.
3. method as claimed in claim 1 or 2, it is characterised in that：It is described that test sample is treated using the excitation source of single wavelength
Parameter setting when product are taken pictures is as follows：
(1) excitation source：470nm or 532nm excitation wavelengths；
(2) filter：535nm, 580nm, 620nm, 660nm or 730nm launch wavelength；
(3) time for exposure：10ms-30s.
4. the method as described in claims 1 to 3 is any, it is characterised in that：It is described to be treated using the excitation source of single wavelength
Parameter setting when test sample product are taken pictures is as follows：
(1) excitation source：470nm excitation wavelengths；
(2) filter：535nm launch wavelengths；
(3) time for exposure：6s.
5. the method as described in claims 1 to 3 is any, it is characterised in that：It is described to be treated using the excitation source of single wavelength
Parameter setting when test sample product are taken pictures is as follows：
(1) excitation source：532nm excitation wavelengths；
(2) filter：620nm launch wavelengths；
(3) time for exposure：15s.
6. the method as described in claim 1 to 5 is any, it is characterised in that：Laser output power when taking pictures is 10%-
100%.
7. the method as described in claim 1 to 6 is any, it is characterised in that：The nematode cyst is browning nematode cyst.
8. any described method of claim 1 to 7 is in plant identification to the application in nematode resistance.
9. a kind of plant identification comprises the following steps to the method for nematode resistance：
(1) by nematode inoculated plant, cultivated；
(2) after completing step (1), taking plant tissue, processing sample obtains sample to be tested, using claim 1 to 7 as sample
In any methods described the nematode in plant tissue is counted；
Resistance of the plant to nematode is judged according to the result of step (2).
10. application as claimed in claim 8, or, the method described in claim 9, it is characterised in that：The nematode is cyst
Nematode.
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Cited By (1)
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| CN113533176A (en) * | 2021-07-09 | 2021-10-22 | 河南省农业科学院 | Method for high-throughput automatic counting of soybean cysts based on fluorescence imaging system |
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