CN103115891A - Method for detecting plasmodiophora brassicae in Chinese cabbages - Google Patents
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Abstract
The invention discloses a method for detecting plasmodiophora brassicae in Chinese cabbages. The method comprises the following steps of: making roots and leaves of the Chinese cabbages into tablets; acquiring infrared spectrograms of the tablets; and analyzing the infrared spectrograms, wherein if the tablets made from the roots have a characteristic peak in at least one part in the ranges of 1748.16+/-0.75cm<-1>, 2851.24+/-0.05cm<-1> and 3013.23+/-0.28cm<-1>, the roots of the Chinese cabbages are infected with the plasmodiophora brassicae; and if the tablets made from the leaves have a characteristic peak in at least one part in the ranges of 1252.54+/-0.50cm<-1> and 1051.98+/-4.23cm<-1>, the leaves of the Chinese cabbages are infected with the plasmodiophora brassicae. According to the method, the plasmodiophora brassicae in the Chinese cabbages can be rapidly and accurately detected in a lossless manner by virtue of a Fourier transform infrared spectroscopy technique, and the detection is convenient to implement, does not depend on skills of people and is high in security and repeatability.
Description
Technical field
The invention belongs to the microorganism detection technical field, relate in particular to a kind of method that detects plasmodiophora brassica bacteria in Chinese cabbage.
Background technology
Food and Agricultural Organization of the United Nations orientates Chinese cabbage one of as most important 20 kinds of vegetables, is important food source.
Plasmodiophora brassica bacteria (Plasmodiophora brassicae) is the obligate parasite in Mastigomycotina (Mastigomycotina), can cause the generation of the crucifer disease-clubroots such as rape, green vegetables, leaf mustard, hot pickled mustard tube, plasmodiophora brassica bacteria also can infect Chinese cabbage, causes the Chinese cabbage clubroot.
Clubroot is the important soil-borne disease of crop in cruciferae, can form on the plant taproot and lateral root vary in size, tumour that shape differs.Can affect the growth that plant shoot divides after root is injured, make the leaf look thin out, growth retardation is downgraded, and fall ill and can cause plant to wilt in flakes and death when serious.This pathogen survives the winter in soil with resting spore, can be more than Survival for 10 Years, and can be through soil, current, seed dispersal, with the root hair of zoospore invaded plants.
In recent years, clubroot harm increases the weight of year by year, and each large growing area of Southwestern China, Central China, northeast and East China all occurred fairly large popular.Clubroot has become the crop in cruciferae Major Diseases, approximately 6,000 ten thousand mu of hazard areas, and the average output loss reaches 20% ~ 30%, and serious field production loss reaches more than 60%, even total crop failure.Cause approximately 1,000,000,000 yuan of rape economic losses, vegetables economic loss approximately 20,000,000,000 every year, greatly restricted sustainable and stable development and the increasing peasant income of oil plant and vegetables industry.For the control of clubroot, still lack at present efficient, low toxicity, low-residual, chemical agent cheaply, and the biological prevention and control agent quantity that effectively controls clubroot also seldom, and easily be subjected to the impact of environmental baseline; Aspect cultural control, to the research of clubroot inadequate system, comprehensively, there are many drawbacks.Therefore, find the cause of disease early, the incidence of grasping clubroot is most important for prevention, monitoring, the control of clubroot.
At present, the detection of plasmodiophora brassica bacteria still mainly depends on traditional Symptom Observation, cause of disease is differentiated, not only takes time and effort, and usually depends on people's micro-judgment, if need accurately identification disease not a duck soup, and required stripped plant tissue sample size is large.PCR (PCR) can be detected the pathogen in plant sample and soil sample, easy to operate, simplified to a certain extent the detection of plasmodiophora brassica bacteria, but need the nucleic acid extraction process, time is longer, and conventional PCR diagnosis often is subjected to false positive and false-negative the impact, and repeatability and accuracy are relatively poor, therefore, be necessary to find a kind of alternative method to judge that accurately whether pathogen exists.
Ftir Spectroscopy (FT-IR) has been widely used in the various fields such as chemistry, agricultural, bio-science, medicine as a kind of instrument of working sample molecular structure, be the effective means of amalyzing substances structure, obtained swift and violent development after 20 century 70s rise.Since the nineties, utilize the FT-IR technology to microorganism classify, the research of Identification and detection also quietly rises.The structure and composition information of the cellular macromolecule materials such as nucleic acid, protein, lipid, carbohydrates can be reflected on infrared spectrogram.Although, the biochemical substances that forms bacterial cell is basic identical, but the difference of some functional group wherein can become the basis of distinguishing and identifying different strains, with this as a kind of novel method (Dai Qunwei that identifies the quick nondestructive that detects microorganism, Dong sends out the .FTIR technology diligent in the application [J] of a few strain Bacteria Identification. Xinan Science and Technology Univ.'s journal, 2009,24 (1): 114-117).Liu Gang etc. utilize the FT-IR technology successfully to differentiate the variety classes (Liu Gang of edible fungi; Liu Jianhong; Deng. the Fourier transform infrared spectroscopy of edible fungi is identified [J]. spectroscopy and analysis spectrum .2004; 24 (8): 941-945); different parts and the different place of production of same edible fungi have been distinguished; kind Yun Xiang etc. compare (Ci Yunxiang to several microorganism infrared spectrums; the Zang Kai match; Deng. the infrared spectroscopic study of several microorganisms [J]. SCI; 2002,23 (6): 1047-1049).At present not yet for there being FT-IR to be applied to the relevant report that plasmodiophora brassica bacteria detects,
Summary of the invention
The invention provides a kind of method that detects plasmodiophora brassica bacteria in Chinese cabbage, use Ftir Spectroscopy, realized quick, harmless, the detection accurately of plasmodiophora brassica bacteria in the Chinese cabbage.
A kind of method that detects plasmodiophora brassica bacteria in Chinese cabbage comprises:
(1) get root or the leaf of Chinese cabbage, make compressing tablet;
(2) gather the infrared spectrogram of described compressing tablet;
(3) infrared spectrogram is analyzed:
If make the infrared spectrogram of compressing tablet at 1748.16 ± 0.75cm by root
-1, 2851.24 ± 0.05cm
-1With 3013.23 ± 0.28cm
-1In scope, characteristic peak appears at least one place, illustrates that the root of Chinese cabbage infects plasmodiophora brassica bacteria;
If make the infrared spectrogram of compressing tablet at 1252.54 ± 0.50cm by leaf
-1With 1051.98 ± 4.23cm
-1In scope, characteristic peak appears at least one place, illustrates that the leaf of Chinese cabbage infects plasmodiophora brassica bacteria.
After the pathogen infection host plant, the nucleic acid of host plant, the metabolism of albumen change, and some secondary metabolites of dynamic accumulation, relevant enzymes etc., simultaneously, the material of pathogen self forms also has difference with host plant, the structure and composition information of these difference materials can be reflected on infrared spectrogram, forms characteristic peak.Infected the root of Chinese cabbage at 1748.16 ± 0.75cm by plasmodiophora brassica bacteria
-1(refer at 1748.16cm
-1Float ± 0.75cm the left and right in the place
-1, other the like), 2851.24 ± 0.05cm
-1With 3013.23 ± 0.28cm
-1Scope has characteristic peak, is infected the leaf of Chinese cabbage at 1252.54 ± 0.50cm by plasmodiophora brassica bacteria
-1, 1051.98 ± 4.23cm
-1Have characteristic peak, and the root of healthy Chinese cabbage or leaf do not have characteristic peak in above-mentioned arbitrary wavelength band.Therefore, according to the infrared spectrogram of root or the leaf of Chinese cabbage to be detected, observe characteristic peak, can determine whether the root of Chinese cabbage or leaf infect plasmodiophora brassica bacteria.
When the compressing tablet of being made by root or leaf is carried out the collection of infrared spectrogram, gather wavelength band and can be 4000 ~ 400cm
-1
For making detection speed faster, preferred,
If make compressing tablet by root, the collection wave band of infrared spectrogram is 1700 ~ 3100cm
-1
If make compressing tablet by leaf, the collection wave band of infrared spectrogram is 1000 ~ 1300cm
-1
Described infrared spectrogram can be gathered by the ftir analysis instrument.
Before infrared spectrogram gathers, need elder generation for the preparation of the root of the Chinese cabbage of spectral analysis or the compressing tablet of leaf, concrete grammar is:
With grind into powder after the root of Chinese cabbage or leaf freeze drying, with the potassium bromide mixing, be pressed into compressing tablet.
Grain diameter influence's infrared spectrum plot quality of the root of Chinese cabbage or leaf powder, the particle diameter of the root of described Chinese cabbage or leaf powder are less than 2 μ m.Particle diameter is excessive, and the compressing tablet light transmission of preparation is poor, and scattering process is strong, easily causes the drift of infrared spectrogram baseline, the bands of a spectrum distorted.
Obvious, the particle diameter of potassium bromide is less than 2 μ m.
For avoiding moisture on the impact of Infrared spectroscopy, before mixing, potassium bromide answers drying to dewater.
The weight ratio of the root of described Chinese cabbage or leaf powder and potassium bromide is 1:100 ~ 3:100, is preferably 1:100.
Potassium bromide can dilute root or the leaf powder of Chinese cabbage, and is easy to the compressing of compressing tablet.The weight ratio of the root of Chinese cabbage or leaf powder and potassium bromide maintains suitable scope, be conducive to comprehensive acquisition characteristics peak-to-peak signal, both can prevent the root of Chinese cabbage or the absorption saturated phenomenon that the leaf powder too much causes, and also can avoid the loss of the characteristic peak of the root of Chinese cabbage or the very few weak signal that causes of leaf powder.
For obtaining high-quality infrared spectrogram, the thickness of described compressing tablet is wanted uniformity, and general, the thickness of described compressing tablet is 0.5 ~ 1mm.
When gathering infrared spectrogram, scanning times is 32 ~ 128 times, and preferred, described scanning times is 32 times.
Suitable scanning times can strengthen detected sample in the information of infrared region, weakens ground unrest, improves spectral signal-noise ratio.
When gathering infrared spectrogram, scanning resolution is 0.125 ~ 32cm
-1, preferred, described scanning resolution is 4cm
-1Resolution is too high, and sweep velocity is slow, affects detection speed; Resolution is too low, can cause the loss of detected sample information, the accuracy that impact detects.
In the gatherer process of described infrared spectrogram, usually need to pass into continuously dry air to the sample area of described ftir analysis instrument, in order to drive away water vapor, avoid the interference of water vapor.
Compared with prior art, beneficial effect of the present invention is:
The present invention detects the method for plasmodiophora brassica bacteria in Chinese cabbage, use Ftir Spectroscopy, can be fast, harmless, detect plasmodiophora brassica bacteria in Chinese cabbage accurately, easy to operate, do not rely on people's experience, and security, repeated high is for effectively prevention and control Chinese cabbage clubroot provide new technological means.
Description of drawings
Fig. 1 for the root of the Chinese cabbage that infected by plasmodiophora brassica bacteria at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 2 is that the root of healthy Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 3 for the leaf of the Chinese cabbage that infected by plasmodiophora brassica bacteria at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 4 is that the leaf of healthy Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 5 is cabbage samples D1, W1, S1 and S2 Genomic PCR amplification electrophoretogram;
Wherein, the size of Marker (DL2000) is followed successively by 100bp, 250bp, 500bp, 750bp, 1000bp and 2000bp
Fig. 6 is that the root sample D1 of Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 7 is that the root sample W1 of Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 8 is that the root sample S1 of Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band;
Fig. 9 is that the leaf sample S2 of Chinese cabbage is at 4000 ~ 500cm
-1The infrared spectrogram of wavelength band.
Embodiment
Below in conjunction with specific embodiment, the present invention is done further explaination.
The preparation of embodiment 1 sample
Cabbage samples gathers the Xinmin City from the LiaoNing, China province subordinate of Shenyang City.
Cabbage samples: the root that infects the Chinese cabbage of plasmodiophora brassica bacteria;
The root (referring to not infect plasmodiophora brassica bacteria) of healthy Chinese cabbage;
Infect the leaf of the Chinese cabbage of plasmodiophora brassica bacteria;
The leaf (referring to not infect plasmodiophora brassica bacteria) of healthy Chinese cabbage.
The basic discriminating of the Chinese cabbage of being infected by plasmodiophora brassica bacteria and judging based on the Symptom Observation of routine and the application of specific PCR experimental technique (R.Faggian S.Parsons.Arapid diagnostic test for clubroot[J] .Horticulture Australia).
(1) with cabbage samples after-70 ℃ of lyophilizations, be placed in mortar, grind to form particle diameter less than the powder of 2 μ m with pestle;
The potassium bromide that (2) will be divided in centrifuge tube is placed in the freeze dryer inner drying, after drying, gets the 100mg potassium bromide and 1mg cabbage samples powder is placed in centrifuge tube, mixes;
(3) with above-mentioned potpourri with 100kg/cm
2The compressing tablet that thickness is the thin plate-like of 0.5 ~ 1mm is made in pressure compacting (1200psi) approximately 8 minutes.
Collection and the data analysis of embodiment 2 Fourier transform infrared spectroscopies (FTIR)
Scan with the compressing tablet of ftir analysis instrument to embodiment 1 preparation, obtain the infrared spectrum of cabbage samples, the cabbage samples that the comparative analysis plasmodiophora brassica bacteria infects and the infrared spectrum of healthy cabbage samples.In scanning process, the sample area in Fourier's infrared spectrometric analyzer is continuous passes into dry air, to drive water vapor.
The Infrared spectrum scanning condition is as follows:
Temperature: room temperature (25 ℃);
Sweep limit: 4000 ~ 500cm
-1
Resolution: 4cm
-1
Scanning times: 100 times.
The analysis of ir data obtains after being analyzed by Grams32@software (Galactic Industries, Salem, NH, USA), analyzes the i.e. 4000 ~ 500cm of spectral range
-1Section.
The position of absorption peak is definite by the central point of example weight, and the mean value that spectral analysis obtains is based on collimation test, baseline calibration, normalization data, can record on this basis the spectral range of absorption peak by same software.Although the average of spectral analysis and the adjustment of normalization can only characterize from vision and provide difference, the factors such as the parameters of spectrum, the calculating of average, statistical study, baseline calibration all can be taken into account.
Table 1 is compared by the main absorption peak of the root sample (n=3) of plasmodiophora brassica bacteria infection and healthy Chinese cabbage
Annotate: A represents absorbance, and v represents stretching vibration, and δ represents flexural vibrations.
As seen from Figure 1, Figure 2, at 4000 ~ 500cm
-1In scope, the root sample that infects the Chinese cabbage of plasmodiophora brassica bacteria has 7 identical peak positions with the root sample of healthy Chinese cabbage, is respectively 1032.69cm
-1, 1240.97cm
-1, 1372.10cm
-1(1381.75cm
-1), 1412.60cm
-1, 1642.09cm
-1(1632.45cm
-1) and 2922.59cm
-1, their absorption band belongs to respectively the stretching vibration of S=O, the flexural vibrations of C-H, the stretching vibration of N=O, the flexural vibrations of O-H, C=C stretching vibration, C-H stretching vibration.In addition, 1748.16cm
-1, 2851.24cm
-1And 3013.23cm
-1Peculiar for the root sample that is subjected to the Chinese cabbage that plasmodiophora brassica bacteria infects, they belong to respectively the stretching vibration of C=O, the stretching vibration of N-H, the stretching vibration of C-H.
Through the repeatedly test of different samples, as shown in Table 1, be subjected to the root sample of the Chinese cabbage that plasmodiophora brassica bacteria infects at 1748.16 ± 0.75cm
-1, 2851.24 ± 0.05cm
-1With 3013.23 ± 0.28cm
-1Have characteristic peak in scope, and the root sample of healthy Chinese cabbage is at 1512.88 ± 0.21cm
-1With 1732.73 ± 0.67cm
-1Has characteristic peak in scope.
Wherein, 1748.16 ± 0.75cm
-1Be illustrated in 1748.16cm
-1Float the left and right in the place, 0.75cm floats
-1, the rest may be inferred for other.
Table 2 is compared by the main absorption peak of the leaf sample (n=3) of plasmodiophora brassica bacteria infection and healthy Chinese cabbage
Annotate: A represents absorbance, and v represents stretching vibration, and δ represents flexural vibrations.
By Fig. 3, Fig. 4 as can be known, at 4000 ~ 500cm
-1In scope, the leaf sample that infects the Chinese cabbage of plasmodiophora brassica bacteria has 4 identical peak positions with the leaf sample of healthy Chinese cabbage, and they are respectively 1412.60cm
-1, 1611.23cm
-1, 2922.59cm
-1And 3402.78cm
-1(3381.57cm
-1), their absorption band belongs to respectively the O-H flexural vibrations, the flexural vibrations of N-H, the stretching vibration of C-H, O-H stretching vibration.In addition, 1051.98cm
-1And 1252.54cm
-1Peculiar by the Chinese cabbage leaf sample that infected by plasmodiophora brassica bacteria, their absorption band belongs to respectively the stretching vibration of S=O, the flexural vibrations of C-H.
Through the repeatedly test of different samples, as shown in Table 2, be subjected to the Chinese cabbage leaf sample of plasmodiophora brassica bacteria infection at 1252.54 ± 0.50cm
-1, 1051.98 ± 4.23cm
-1Have characteristic peak in scope, and the leaf sample of healthy Chinese cabbage is at 1092.48 ± 0.41cm
-1Has characteristic peak in scope.
Embodiment 3 utilizes the plasmodiophora brassica bacteria in PCR (PCR) and Ftir Spectroscopy (FTIR) detection Chinese cabbage
Choose root sample (D1) and the uncertain cabbage samples that whether infects (root sample W1, the S1 of known infection plasmodiophora brassica bacteria (P.brassicae); Leaf sample S2), use the method that ISOPLANT II kit (Nippon Gene Co.Ltd) provides to specifications to extract Chinese cabbage tissue gene group DNA, utilize the Auele Specific Primer of plasmodiophora brassica bacteria to carry out pcr amplification, amplified production is carried out 1% agarose gel electrophoresis analysis.
Auele Specific Primer is:
NS7 (upstream primer): 5 '-GAGGCAATAACAGGTCTGTGATGC-3 ';
Pb3R (downstream primer): 5 '-ACCATACCCAGGGCGATTG-3 '.
Pcr amplification system such as following table.
| Composition | Content (uL) |
| |
1 |
| 10×buffer | 2 |
| DNTP potpourri (each 2.5mmol/L) | 1.5 |
| NS7(20umol/L) | 0.5 |
| Pb3R(20umol/L) | 0.5 |
| TaqDNA polymerase (1U/uL) | 1 |
| Ultrapure water | 13.5 |
| Amount to | 20 |
Reaction conditions:
95 ℃ of denaturation 4min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, amount to 30 circulations; 72 ℃ are extended 10min.
As shown in Figure 5, utilize clubroot Auele Specific Primer NS7, after Pb3R carried out the PCR specific amplification, sample D1, S1, S2 can obtain size and be the specific band of 507bp, and sample W1 does not obtain specific band.Therefore, sample D1, S1, S2 are subject to the infection of plasmodiophora brassica bacteria, and sample W1 is not subject to the infection of plasmodiophora brassica bacteria.
To sample D1, S1, S2, W1 prepares compressing tablet according to the method for embodiment 1, gathers Fourier transform infrared spectroscopy figure according to the method for embodiment 2 and goes forward side by side line number according to one's analysis.
Table 3 a sample D1, S1, the W1 characteristic peak is analyzed
As shown in Fig. 6, Fig. 7, Fig. 8 and table 3, at 4000 ~ 500cm
-1In scope, although sample D1 is at 1742.16cm
-1, 2854.22cm
-1, 3012.65cm
-1Characteristic peak not in the fluctuation range of old complaint characteristic peak, but owing to there being systematic error, so think that above-mentioned three characteristic peaks of sample D1 are the old complaint characteristic peak, in like manner, think that sample S1 is at 2854.03cm
-1, 3011.93cm
-1Characteristic peak be the old complaint characteristic peak, and sample W1 does not have the old complaint characteristic peak.Therefore, can judge that sample D1, S1 are infected by plasmodiophora brassica bacteria, sample W1 is not infected by plasmodiophora brassica bacteria.
As shown in Fig. 9 and table 4, at 4000 ~ 500cm
-1In scope, consider systematic error, leaf sample S2 is at 1054.41cm
-1, 1255.17cm
-1Has disease leaf characteristic peak.Therefore, can judge that sample S2 is infected by plasmodiophora brassica bacteria.
Table 4 leaf sample S2 characteristic peak is analyzed
Annotate: A represents absorbance, and v represents stretching vibration, and δ represents flexural vibrations.
The Ftir Spectroscopy that obtains thus (FTIR) qualification result is consistent with PCR (PCR) qualification result, illustrates whether method of the present invention can infect plasmodiophora brassica bacteria by the precise Identification Chinese cabbage.
Claims (7)
1. a method that detects plasmodiophora brassica bacteria in Chinese cabbage, is characterized in that, comprising:
(1) get root or the leaf of Chinese cabbage, make compressing tablet;
(2) gather the infrared spectrogram of described compressing tablet;
(3) infrared spectrogram is analyzed:
If make the infrared spectrogram of compressing tablet at 1748.16 ± 0.75cm by root
-1, 2851.24 ± 0.05cm
-1With 3013.23 ± 0.28cm
-1In scope, characteristic peak appears at least one place, illustrates that the root of Chinese cabbage infects plasmodiophora brassica bacteria;
If make the infrared spectrogram of compressing tablet at 1252.54 ± 0.50cm by leaf
-1With 1051.98 ± 4.23cm
-1In scope, characteristic peak appears at least one place, illustrates that the leaf of Chinese cabbage infects plasmodiophora brassica bacteria.
2. the method for plasmodiophora brassica bacteria in detection as claimed in claim 1 Chinese cabbage, is characterized in that, the preparation method of described compressing tablet is: with grind into powder after the root of Chinese cabbage or leaf freeze drying, with the potassium bromide mixing, be pressed into compressing tablet.
3. the method for plasmodiophora brassica bacteria in detection Chinese cabbage as claimed in claim 2, is characterized in that, the particle diameter of the root of described Chinese cabbage or leaf powder is less than 2 μ m.
4. the method for plasmodiophora brassica bacteria in detection Chinese cabbage as claimed in claim 2, is characterized in that, the weight ratio of the root of described Chinese cabbage or leaf powder and potassium bromide is 1:100 ~ 3:100.
5. the method for plasmodiophora brassica bacteria in detection Chinese cabbage as claimed in claim 2, is characterized in that, the thickness of described compressing tablet is 0.5 ~ 1mm.
6. the method for plasmodiophora brassica bacteria in detection Chinese cabbage as claimed in claim 1, is characterized in that, the collection wave band of described infrared spectrogram is 4000 ~ 400cm
-1
7. the method for plasmodiophora brassica bacteria in detection Chinese cabbage as claimed in claim 6, is characterized in that,
If make compressing tablet by root, the collection wave band of infrared spectrogram is 1700 ~ 3100cm
-1
If make compressing tablet by leaf, the collection wave band of infrared spectrogram is 1000 ~ 1300cm
-1
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| CN106124479A (en) * | 2016-09-05 | 2016-11-16 | 海南大学 | A kind of method utilizing situ Raman Spectroscopy Non-Destructive Testing chrysanthemum Phyllostachys pubescens |
| CN107917889A (en) * | 2017-11-20 | 2018-04-17 | 包头东宝生物技术股份有限公司 | Using the method for examination of infrared spectrum gelatin sample |
| CN108645808A (en) * | 2018-04-17 | 2018-10-12 | 云南中烟工业有限责任公司 | One grows tobacco early blossoming seedling stage determination method |
| CN108693131A (en) * | 2018-04-17 | 2018-10-23 | 云南中烟工业有限责任公司 | One grows tobacco yellows determination method |
| CN108693131B (en) * | 2018-04-17 | 2023-12-01 | 云南中烟工业有限责任公司 | Tobacco yellowing disease judging method |
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