Application of proline derivative-rhodamine B conjugate as fluorescent tracer in crop and weed recognition

Abstract

The invention belongs to the technical field of fluorescent tracer agent application, and particularly relates to application of a proline derivative-rhodamine B conjugate as a fluorescent tracer agent in crop and weed recognition. The structural formula of the proline derivative-rhodamine B conjugate is as follows: after the proline derivative-rhodamine B conjugate obtained by the invention is directionally applied to crop plants as a fluorescent tracer, the crop plants can be labeled by fluorescence, and a machine vision system is assisted to distinguish crops and weeds; precise pesticide spraying or mechanical weeding is carried out according to spatial distribution of weeds, automatic and intelligent weed control is achieved, and the usage amount of herbicide is reduced.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 202510372212.0, the application date of 27 March 2025, and the invention title of "Amino acid derivative-rhodamine B conjugate and its preparation method and application as a fluorescent tracer". TECHNICAL FIELD

[0002] The present application belongs to the technical field of fluorescent tracer application, and specifically relates to the application of proline derivative-rhodamine B conjugate as a fluorescent tracer in crop and weed identification. BACKGROUND

[0003] In the process of crop growth, weeds, as a kind of harmful organisms that harm farmland, compete with crops for resources such as sunlight, water, air, and nutrients, greatly affecting the growth and yield of crops. At present, the common methods for farmland weed control include physical control, chemical control, biological control, and agricultural control. Among them, chemical control is widely used in weed control due to its high efficiency, economy, and labor-saving advantages. However, the unreasonable use of herbicides has also caused environmental pollution, pesticide damage, weed resistance, and pesticide residues, etc. Therefore, improving the effective utilization rate of pesticides, reducing the use of pesticides, and developing intelligent plant protection machinery and application technology are the only way for the development of agricultural modernization plant protection. Among them, using modern information technology to distinguish farmland crops and weeds, and clearly defining the spatial distribution of weeds, and then applying precise pesticide or mechanical weeding to weeds, is of great significance for effectively reducing the amount of herbicide used and achieving intelligent weed control in farmland. Currently, machine vision technology is mainly used to identify crops and weeds.

[0004] Machine vision technology uses deep learning and other methods to establish a database of crop and weed morphological characteristics, and then identifies crops / weeds. It has the advantages of being fast, low-cost, and high-resolution. However, the existing technology is suitable for row-by-row weed control, and the recognition degree of inter-plant weeds is not high enough. In addition, in complex situations (such as high weed density conditions), the recognition degree is low, and a large amount of weed and crop characteristic database needs to be established, which is tedious in the early stage. In addition, as plants grow, the morphological characteristics of crops and weeds also change, further increasing the difficulty of plant morphological feature extraction.

[0005] Crop plant signal technology applies exogenous fluorescent markers to crops through seed treatment and other means, so that crops are labeled with fluorescent substances, while weeds do not contain markers. The characteristic spectrum of the marker can be captured by a CCD camera to accurately distinguish crops from weeds. Therefore, crop plant signal technology helps to improve the efficiency and accuracy of automatic weed control in farmland, and can establish a high spatial resolution crop / weed precise positioning distribution map in real time. Compared with computer learning algorithm-based vision technology, crop plant signal technology has the advantages of simplicity and accuracy.

[0006] Fluorescent markers are the core and basis of crop plant signal technology. Previous studies have shown that the fluorescent tracers that can be absorbed and conducted by plants are mainly coumarin (such as coumarin 120) and rhodamine (such as rhodamine B, Rh-B) (Wang, Z.; Amirkhani, M.; Avelar, S. A. G.; Yang, D.; Taylor, A. G. Systemic Uptake of Fluorescent Tracers by Soybean (Glycine max (L.) Merr.) Seed and Seedlings. Agriculture 2020, 10, 248. https: / / doi.org / 10.3390 / agriculture10060248). Further studies have shown that rhodamine B has unique fluorescence characteristics (λex / λem = 555 / 582 nm), which are highly distinguishable from the fluorescence characteristics of plant chlorophyll. Rhodamine B can be distinguished from chlorophyll under relatively wide fluorescence conditions (λex = 535 nm, λem = 580-582 nm) and can be used for fluorescence imaging detection. Compared with other fluorescent tracers, rhodamine B has the advantages of low cost, strong light stability, high fluorescence yield, wide wavelength range, wide pH adaptation range, good cell membrane permeability, and no toxic side effects on cells, and therefore has more application prospects in plant tracers.

[0007] Studies have found that rhodamine B can be absorbed by bean and soybean seeds and penetrate into the interior of the seeds, or be absorbed by the root systems of celery, lettuce, and tomatoes and conducted upward through the plant xylem to the upper part of the seedlings. However, overall, rhodamine B is mainly distributed in the hypocotyls of plants (soybeans and beans), and the fluorescence intensity of rhodamine B in the epicotyls is 35% (beans) and 11% (soybeans) of that in the hypocotyls, and the fluorescence intensity of rhodamine B in the leaves is only about 8% of that in the hypocotyls, indicating that the upward conducting ability of rhodamine B is weak, and the internal absorption and conducting performance of the tracer needs to be further improved.

[0008] In view of the insufficient internal absorption and conducting performance of the existing tracers, which limits the capture of fluorescent signals by machines, the structure of the existing tracer rhodamine B is optimized to improve its internal absorption and conducting performance, and a fluorescent tracer for precise weeding technology is developed. SUMMARY

[0009] The purpose of the present application is to overcome the defects of the prior art, and to propose the application of proline derivative-rhodamine B conjugate as a fluorescent tracer in crop and weed identification. The present application optimizes the structure of the existing fluorescent marker rhodamine B, introduces an amino acid fragment into the molecular structure of rhodamine B, and develops a fluorescent marker suitable for crop plant signal technology.

[0010] To achieve the above object, the present application adopts the following technical solutions:

[0011] The amino acid derivative-Rhodamine B coupling compound has the following structural formula:

[0012]

[0013] The coupling bond in the above structure is an amide bond formed by the amine group of the amino acid derivative and the carboxyl group of Rhodamine B.

[0014] The group R is selected from one of

[0015] Further preferably, the group R is selected from

[0016] The present application also provides a preparation method of the above-mentioned amino acid derivative-Rhodamine B coupling compound.

[0017] The present application further provides the use of the above-mentioned amino acid derivative-Rhodamine B coupling compound as a fluorescent tracer.

[0018] Preferably, the use of the amino acid derivative-Rhodamine B coupling compound as a fluorescent tracer in crop and weed identification.

[0019] Further preferably, the crop is soybean, corn and tobacco.

[0020] The amino acid derivative-Rhodamine B coupling compound in the present application includes the coupling compound formed by the amino acid methyl ester and Rhodamine B, and the coupling compound formed by the acidified amino acid and Rhodamine B.

[0021] Compared with the prior art, the present application has the beneficial effects that:

[0022] After the amino acid derivative-Rhodamine B coupling compound obtained by the present application is used as a fluorescent tracer, the content of the tracer in the aboveground part of the crop plant is significantly increased, which is beneficial for the crop-weed identification by the machine vision system, and precise pesticide spraying or mechanical weeding can be carried out according to the spatial distribution of weeds, realizing the automation and intelligence of weed control, significantly reducing the use amount of herbicide, effectively reducing the influence of chemical herbicide on the ecological environment, reducing the occurrence of herbicide damage, delaying the development of drug resistance, and reducing the risk of pesticide residues. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a fluorescence detection diagram in a soybean plant;

[0024] Figure 2 is a fluorescence detection diagram in a corn plant;

[0025] Figure 3 ​is the fluorescence detection chart in tobacco plants;

[0026] Figure 4 is the standard curve of three fluorescence tracers;

[0027] Figure 5 is the content chart of the fluorescence tracer in different parts of soybean;

[0028] Figure 6 is the content chart of the fluorescence tracer in different parts of corn;

[0029] Figure 7 is the content chart of the fluorescence tracer in different parts of tobacco;

[0030] Figure 8 is the distribution proportion chart of the fluorescence tracer in soybean plants;

[0031] Figure 9 is the distribution proportion chart of the fluorescence tracer in corn plants;

[0032] Figure 10 is the distribution proportion chart of the fluorescence tracer in tobacco plants;

[0033] Figure 11 is the effect chart of three fluorescence tracers on the growth of soybean plants;

[0034] Figure 12 is the effect chart of three fluorescence tracers on the growth of corn plants;

[0035] Figure 13 is the effect chart of three fluorescence tracers on the growth of tobacco plants;

[0036] Figure 14 is the schematic diagram of the seed sowing position of crops and weeds;

[0037] Note: Figure 5 , 6 , 7, 11, 12, 13 Different letters in the treatment difference (P < 0.05). DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be described in detail below in combination with the drawings and examples.

[0039] Example 1

[0040] Rhodamine B and amino acid methyl ester (amino acid methyl ester general formula ) as raw materials, through amidation and ester hydrolysis to construct amino acid derivative-Rhodamine B coupling compound, the specific synthesis route is as follows reaction formula:

[0041]

[0042] wherein, amidation condition a: N-methylmorpholine (NMM), l-ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI), DMAP, anhydrous dichloromethane, r.t (room temperature);

[0043] ester hydrolysis condition b: lithium hydroxide, THF / H20 (V / V = 2:1), r.t (room temperature);

[0044] amino acid methyl ester general formula wherein, R is determined according to the selected amino acid, such as the amino acid selected from the group consisting of isoleucine, tyrosine, tryptophan, proline, alanine.

[0045] For example, proline methyl ester hydrochloride (2.48 g, 15 mmol) and N- methylmorpholine (NMM) (2.20 mL, 20 mmol) were dissolved in anhydrous dichloromethane (150 mL). Then the reaction mixture was cooled to 0 °C, and rhodamine B (4.79 g, 10 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI) (2.88 g, 15 mmol), 4-dimethylaminopyridine (DMAP) (0.12 g, 1 mmol) were added successively. The resulting solution was stirred at room temperature for 3 h. After the reaction was completed, water was added, and the organic layer was extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution alternately for 2-3 times, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 2 (proline methyl ester-rhodamine B conjugate, conjugate 7 in Table 1) was obtained by column chromatography.

[0046] Compound 2 (3.22 g, 5 mmol) was dissolved in a mixture of water (10 mL) and THF (20 mL), and lithium hydroxide (0.63 g, 15 mmol) was added under ice bath. The reaction was slowly warmed to room temperature and continued for 1 h. After the reaction was completed, it was acidified with 1 M hydrochloric acid solution to pH = 2, and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution alternately for 2-3 times, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 3 (proline-rhodamine B conjugate, conjugate 8 in Table 1) was obtained by column chromatography.

[0047] Isoleucine methyl ester-rhodamine B conjugate (conjugate 1 in Table 1), isoleucine-rhodamine B conjugate (conjugate 2 in Table 1), tyrosine methyl ester-rhodamine B conjugate (conjugate 3 in Table 1), tyrosine-rhodamine B conjugate (conjugate 4 in Table 1), tryptophan methyl ester-rhodamine B conjugate (conjugate 5 in Table 1), tryptophan-rhodamine B conjugate (conjugate 6 in Table 1), proline methyl ester-rhodamine B conjugate (conjugate 7 in Table 1), proline-rhodamine B conjugate (conjugate 8 in Table 1), alanine methyl ester-rhodamine B conjugate (conjugate 9 in Table 1), and alanine-rhodamine B conjugate (conjugate 10 in Table 1) were synthesized according to the above method.

[0048] The above amino acid derivative-rhodamine B conjugates were analyzed by hydrogen spectrum and mass spectrum, and the data are shown in Table 1.

[0049] Table 1 Nuclear magnetic hydrogen spectrum and high resolution mass spectrum data of amino acid derivative-rhodamine B conjugates

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] Example 2

[0056] 1. Qualitative study on the systemic conductive performance of fluorescent tracers in plant seedlings (taking tracers No. 1, 2, 7 and 8 as examples)

[0057] 1.1 Plant seedling culture

[0058] Soybean, corn and tobacco seeds were sowed in 15 cm diameter flowerpots with 250 g substrate at a sowing depth of 2 cm, and the seedlings were cultured until the soybean true leaves were fully unfolded, the first leaf of the corn was perpendicular to the leaf sheath, and the tobacco was at the eight-leaf stage.

[0059] The fluorescent tracers such as rhodamine B or amino acid derivative-rhodamine B conjugates were added to the Hoagland's nutrient solution to prepare a hydroponic solution containing 60 mg / L of the tracer. The cultured seedlings were washed clean and placed in a plastic cup containing 200 mL of the hydroponic solution, and a blank control of Hoagland's nutrient solution was prepared. The culture was carried out at 25°C under a light / dark cycle of 16 h / 8 h for 4 days, and each treatment was repeated 4 times.

[0060] Hogland's nutrient solution preparation method: take 1.26g of Hogland's nutrient solution medium, 0.945g of calcium nitrate, heat and dissolve in 1000ml of distilled water, high pressure sterilization at 115°C for 20min for standby.

[0061] 1.2 Study on the systemicity of fluorescent tracer

[0062] The distribution of fluorescent tracer in different parts of the plant was detected by stereoscopic fluorescence microscope, and whether the tracer could be conducted to the stem and leaf part of the plant was used as the criterion to evaluate its systemicity (compared with rhodamine B). The soybean seedlings treated in 1.1 were divided into three leaf, true leaf, epicotyl, cotyledon, hypocotyl, and root six parts; corn was divided into root, leaf sheath and leaf three parts; tobacco was divided into root, stem and leaf three parts; and RFP detection module (excitation wavelength 541-551nm, detection wavelength 590nm) was used for screening.

[0063] 1.2.1 Evaluation of the systemicity of fluorescent tracer in soybean plants

[0064] The aboveground parts (three leaves, true leaves, epicotyls, cotyledons, and hypocotyls) of soybean plants were observed by stereoscopic fluorescence microscope, and it was found that, as shown in Figure 1 , compared with the control tracer rhodamine B, the fluorescence intensity of tracers 7 and 8 in the aboveground parts of soybean plants was increased, and the fluorescence intensity of tracer 2 in the true leaf part was higher than that of rhodamine B, indicating that the coupling with amino acids such as proline could improve the systemicity of rhodamine B in soybean plants, and isoleucine could enhance the transmission ability of rhodamine B in the leaf part to some extent.

[0065] 1.2.2 Evaluation of the systemicity of fluorescent tracer in corn plants

[0066] The fluorescence of corn plants was detected by stereoscopic fluorescence microscope, and it was found that, as shown in Figure 2 , compared with the control tracer rhodamine B, the fluorescence intensity of tracers 1, 2, 7 and 8 in the aboveground parts (leaves and leaf sheaths) of corn plants was increased, indicating that the coupling with amino acids such as isoleucine and proline could improve the systemicity of rhodamine B in corn plants.

[0067] 1.2.3 Evaluation of the systemicity of fluorescent tracer in tobacco plants

[0068] The tracer-treated tobacco seedlings were detected under stereoscopic fluorescence microscope. As shown in Figure 3 , compared with the control tracer rhodamine B, the fluorescence intensity of tracers 1, 7 and 8 in the aboveground parts (leaves and stems) of tobacco plants was increased, indicating that the coupling with amino acids such as isoleucine and proline could improve the systemicity of rhodamine B in tobacco plants.

[0069] In general, the fluorescence intensity of amino acid derivative-rhodamine B conjugates in the aboveground parts of plants was higher than that of rhodamine B, indicating that isoleucine, proline, and other amino acids had obvious effects on the systemic conductive properties of rhodamine B in plants, especially in monocotyledonous plants (corn).

[0070] 2 Quantitative analysis of fluorescent tracers in different parts of plants

[0071] 2.1 Test method

[0072] Using 7 and 8 as representatives, the content of fluorescent tracers in different parts of plants was further detected by liquid chromatography.

[0073] (1) Sample treatment

[0074] 0.5 g of each part of soybean, corn, and tobacco seedlings treated with rhodamine B, 7, and 8 in 1.1 was ground and added with 4 mL of acetonitrile, and then ultrasonically extracted for 40 min. After 1 day of extraction, the sample was concentrated to 1 mL and filtered through a 0.22 μm organic microporous filter for high-performance liquid chromatography analysis to determine the content of fluorescent tracers in different parts of soybean, corn, and tobacco.

[0075] Liquid chromatography detection conditions: C 18 The chromatographic column (C18-AQ, 5 μm, 250 mm x 4.6 mm) was used, the detection wavelength was 560 nm, the injection volume was 10 μL, the flow rate was 1 mL / min, and the column temperature was 35°C. The mobile phase was 0.1% formic acid aqueous solution and acetonitrile, and the specific ratio was as shown in Table 2:

[0076] Table 2 Proportion of mobile phase for three tracers

[0077]

[0078]

[0079] (2) Preparation of standard solution and drawing of standard curve

[0080] 5.0 mg of rhodamine B standard, 7, and 8 fluorescent tracers were weighed and dissolved in acetonitrile to a constant volume of 10 mL to obtain a 500 mg / L standard stock solution. The standard solution was then diluted with acetonitrile in stages, the absorption peak area of each standard solution was measured, and the standard curve was drawn.

[0081] (3) Recovery rate test

[0082] The blank samples of each part of soybean, corn and tobacco were added with 100.0000, 33.3333, 11.1111 mg / L of standard solution at three levels, and each level was repeated three times. The sample pretreatment and analysis were carried out according to the method, and the addition recovery rate and relative standard deviation were calculated.

[0083] 2.2 Test results

[0084] 2.2.1 Construction and verification of HPLC analysis method

[0085] The standard curve was drawn according to the concentration gradient, and the correlation coefficients of rhodamine B, No. 7 and No. 8 tracers were 0.9996, 0.9990 and 0.9935 respectively. The mass concentration and the corresponding response value showed good linear relationship within the concentration range of the standard curve. Figure 4 As shown in Table 3, the average recovery rates of rhodamine B, No. 7 and No. 8 tracers were between 81% and 109%, and the relative standard deviations of the three were less than 0.65%, indicating that the method met the analysis requirements.

[0086] Table 3 Verification of analysis method of three tracers

[0087]

[0088] 2.2.2 Content of fluorescent tracer in different parts of soybean

[0089] From the distribution of tracers in the aboveground part of soybean (Figure A in Table 3), Figure 5 the content of No. 7 tracer in the hypocotyl, true leaf and three-leaf of soybean was significantly higher than that of rhodamine B, and the content of No. 8 tracer in the true leaf was significantly higher than that of rhodamine B; from the distribution of tracers in the underground part of soybean (Figure B in Table 3), Figure 5 the content of rhodamine B in the root of soybean was significantly higher than that of No. 7 and No. 8 tracers, and there was no significant difference between No. 7 and No. 8, indicating that the absorption capacity of soybean root (underground part) to rhodamine B was stronger, but considering the content of three tracers in the aboveground part of soybean, the content of No. 7 tracer was higher than that of rhodamine B and No. 8 tracer, indicating that the systemic conductive performance of No. 7 tracer in soybean seedlings was significantly improved compared with rhodamine B.

[0090] 2.2.3 Content of fluorescent tracer in different parts of corn

[0091] From the distribution of tracers in different parts of corn plant (Figure A in Table 3), Figure 6, A. shoot; B. root), the content of No. 8 tracer in the leaf sheath and leaf was significantly higher than that of No. 7 tracer and Rhodamine B. The content of No. 8 tracer in the root was significantly higher than that of No. 7 tracer and Rhodamine B, indicating that the absorption capacity of corn root system to No. 8 tracer was the strongest. In general, the content of No. 7 and No. 8 tracers in the shoot of corn was higher than that of Rhodamine B, indicating that the systemic conductive performance of No. 7 and No. 8 tracers in corn seedlings was better than that of the control agent Rhodamine B.

[0092] 2.2.4 Content of fluorescent tracers in different parts of tobacco

[0093] From the distribution of tracers in different parts of tobacco plants Figure 7 , A. shoot; B. root), the content of No. 8 tracer in the leaf sheath and leaf was significantly higher than that of No. 7 tracer and Rhodamine B. The content of No. 8 tracer in the root was significantly higher than that of No. 7 tracer and Rhodamine B, indicating that the absorption capacity of corn root system to No. 8 tracer was the strongest. In general, the content of No. 7 and No. 8 tracers in the shoot of corn was higher than that of Rhodamine B, indicating that the systemic conductive performance of No. 7 and No. 8 tracers in corn seedlings was better than that of the control agent Rhodamine B.

[0094] 2.2.5 Distribution proportion of fluorescent tracers in three plants

[0095] In three crop plants, the proportion of three tracers in the root was the highest. In the proportion distribution of soybean shoot (hypocotyl, cotyledon, epicotyl, true leaf, three-leaf five-part total), No. 7 tracer was the highest, followed by No. 8 tracer, and Rhodamine B was the smallest Figure 8 ); in the proportion distribution of corn plant shoot (leaf sheath and leaf total), No. 7 tracer was higher than Rhodamine B Figure 9 , combined with the results of 2.2.3, the content of No. 7 and No. 8 tracers in the shoot of corn was significantly higher than that of Rhodamine B; in the proportion distribution of tobacco plant shoot (stem and leaf total), the distribution of No. 8 tracer in the shoot was significantly higher than that of No. 7 tracer and Rhodamine B Figure 10 ).

[0096] In general, although the tracers Rhodamine B and No. 7 and No. 8 tracers were mainly distributed in the roots of soybean, corn and tobacco, the content of No. 7 and No. 8 tracers in the shoot of three crop plants was significantly higher than that of Rhodamine B, indicating that the systemic conductive performance of No. 7 and No. 8 tracers in soybean, corn and tobacco was better than that of Rhodamine B, indicating that the introduction of amino acid groups into the Rhodamine B molecule could significantly improve its upward transmission capacity.

[0097] 3 Safety evaluation of fluorescent tracers on plants

[0098] 3.1 Test method

[0099] (1) The effect of fluorescent tracer on the germination of soybean and other three crops: The effect of three tracers on the germination of soybean, corn and tobacco seeds was studied by paper method. 200 mL of rhodamine B, 7 and 8 tracer solutions with concentrations of 3.75 mg / L, 7.50 mg / L, 15.00 mg / L, 30.00 mg / L and 60.00 mg / L were prepared by dissolving in acetonitrile and diluting with deionized water.

[0100] Soybean seeds were soaked in the above solutions for 30 min, and intact seeds were selected, dried in the shade and placed on 2 layers of germination paper, covered with 1 layer of germination paper, 50 seeds per group, repeated 3 times, treated with distilled water as control, cultured in dark at 25°C and humidity of 80%. The number of germinated seeds on the 5th day and the 8th day was recorded according to the requirements of Association of Official Seed Analysts (AOSA).

[0101] Corn seeds were soaked in a dark environment at 25°C for 12 h, washed and dried in the shade, then placed in sterile culture dishes (18 cm) with 2 layers of wet filter paper, 30 seeds per dish, repeated 3 times per group, with distilled water as control. Cultured at 25°C, the number of germinated seeds on the 4th day and the 7th day was recorded according to the requirements of AOSA.

[0102] Full-grain tobacco seeds were selected, soaked in a dark environment at 25°C for 24 h, washed and dried in the shade at 20°C in the dark, treated with distilled water as control. The seeds were placed in 9 cm culture dishes with 2 layers of wet filter paper, incubated at 25°C in a 16 h light and 8 h dark environment, with 3 replicates per treatment and 50 seeds per group. The number of germinated seeds on the 7th day and the 14th day was recorded according to the requirements of AOSA. The germination potential and germination rate were calculated.

[0103] (2) The effect of fluorescent tracer on the growth of soybean and other three crops: The fresh weight of 1.1 treated soybean seedlings was measured, including the fresh weight of true leaves, upper hypocotyls, cotyledons, lower hypocotyls and roots; the fresh weight of corn seedling roots, leaf sheaths and leaves; the fresh weight of tobacco roots, stems and leaves, to study the effect of rhodamine B, 7 and 8 tracers on the growth of seedlings of the three crops under the concentration conditions.

[0104] 3.2 Data processing

[0105] Data statistical analysis was performed using IBM SPSS statistics 25 software, and Duncan's new multiple range method was used for significant difference analysis.

[0106] 3.3 Test results

[0107] 3.3.1 The effect of fluorescent tracer on seed germination

[0108] By comparing the germination energy and germination rate of soybean, corn and tobacco seeds treated with different treatments, it was found that the germination rate and germination rate of some tracer treatments were slightly higher than the blank control, but the germination energy and germination rate of all treatments and the control had no significant difference (Table 4), indicating that each tracer had no obvious effect on the germination of soybean, corn and tobacco seeds.

[0109] Table 4 Effect of different concentrations of three tracers on seed germination

[0110]

[0111] Note: The data in the table are expressed as mean ± standard error, and the same column with different letters represent significant difference (P<0.05)

[0112] 3.3.2 Effect of fluorescent tracer on plant growth

[0113] The fresh weight of each part of soybean, corn and tobacco plants treated with 60 mg / L rhodamine B, No. 7 and No. 8 tracers for 4 days had no significant difference (P>0.05) compared with the blank control (Table 5), indicating that under this concentration treatment, the three fluorescent tracers had no obvious effect on the growth of soybean, corn and tobacco seedlings. Figures 11-13

[0114] In summary, rhodamine B, No. 7 and No. 8 tracers had no obvious effect on the seed germination and seedling growth of soybean, corn and tobacco, indicating that rhodamine B, No. 7 and No. 8 tracers had high safety for soybean, corn and tobacco.

[0115] 4 Verification of the recognition degree of fluorescent tracers between crops and weeds

[0116] Pot culture method was used to test the distribution of fluorescent tracers in crop and weed plants after seed treatment, and to evaluate the recognition degree of fluorescent tracers for crops and weeds.

[0117] The specific method is as follows:

[0118] 4.1 Seed treatment

[0119] The seeds of soybean, corn, tobacco and other crops, and gooseweed, barnyard grass, dogtail grass, amaranth and other weeds were used as test objects. 0.01 g of fluorescent tracer, 0.01 g of adhesive (sodium carboxymethyl cellulose), and 0.48 g of deionized water were mixed uniformly and then attached to the surface of 10 g of crop or weed seeds with a vortex oscillator. The mixture was dried in the room temperature and ready for use. 10 g of crop or weed seeds treated with 0.5 g of deionized water were used as control.

[0120] 4.2 Substrate treatment

[0121] ​The soil retrieved from the teaching farm of Inner Mongolia Agricultural University is dried and sieved (2 mm aperture) to remove large stones and other interfering substances. 1500 g of soil is placed in a flowerpot with an 8 cm diameter, compacted and leveled, and 300 mL of deionized water is added to maintain a soil moisture content of 20%.

[0122] 4.3 Seeding

[0123] One treated crop seed is sown at the center of the flowerpot, and 8 weed seeds (2 of each type) are sown at 2 cm and 4 cm from the crop seed, respectively, with the crop seed as the center. The seeding depth is 3 cm, and 4 pots (4 replicates) are planted for each crop seed. Figure 14 The flowerpots are placed in an artificial climate chamber (25°C, humidity 40%, light / dark: 16 / 8 h) for cultivation. When the corn reaches the 3-4 leaf stage, the soybean reaches the two-leaf stage, and the tobacco reaches the 4-leaf stage, the distribution of the fluorescent tracer in each plant (crop, weed) is detected.

[0124] 4.4 Detection of the distribution of the fluorescent tracer in crops and weeds

[0125] The content of the fluorescent tracer in the roots, stems (sheaths), and leaves of each crop and weed plant is tested by liquid chromatography. The extraction method and testing method are the same as those in "2. Quantitative analysis of the fluorescent tracer in different parts of the plant".

[0126] 4.5 Results

[0127] The distribution of the amino acid derivative-rhodamine B conjugates 7 and 8 and rhodamine B in soybean, corn, tobacco, and four weed seedlings is detected by liquid chromatography. The results are shown in Tables 5-7. It is found that the fluorescent tracer mainly exists in the roots of the crops, and although the content in the aboveground parts (stems and leaves) is relatively lower than that in the roots, it is still significantly distributed in the stems and leaves of the crops and can be used for fluorescence detection. The fluorescent tracer is only detected in the roots of the weeds 2 cm away from the crops (content <0.4 mg / L), and no fluorescent tracer is detected in the aboveground parts of the weeds, indicating that the amino acid derivative-rhodamine B conjugates 7 and 8 and rhodamine B have less lateral migration in the soil, and therefore, the fluorescent tracer developed in the present application has high recognition between crops and weeds.

[0128] Table 5 Content of the fluorescent tracer in different parts of soybean and weeds (mg / L)

[0129]

[0130] Table 6 Content of the fluorescent tracer in different parts of corn and weeds (mg / L)

[0131]

[0132] Table 7 Content of fluorescent tracer in different parts of tobacco and weeds (mg / L)

[0133]

[0134] The above study shows that after hydroponic treatment, the content of amino acid derivative-rhodamine B conjugate in the above-ground parts (stems and leaves) of soybean, corn and tobacco is significantly higher than that of the control agent rhodamine B, especially the 7th and 8th conjugates containing proline, and the upward transmission ability in different plants is significantly higher than that of rhodamine B, indicating that the improved conjugate has more excellent systemic transmission performance, and has no adverse effect on seed germination and seedling growth of plants, and the fluorescence tracer has high discrimination between crops and weeds after seed treatment, and can be used for crop / weed identification.

[0135] The upper and lower limits of the process parameters (such as temperature, time, etc.) of the present application and the interval values can all realize the present method, and examples are not listed here.

[0136] The contents not described in detail in the present application can adopt the conventional technical knowledge in the art.

[0137] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application and are not limited. Although the present application has been described in detail with reference to the examples, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. Use of proline derivative-rhodamine B conjugates as fluorescent tracers in crop and weed discrimination; characterized in that, The crop is soybean, corn and tobacco; The structure of the proline derivative-Rhodamine B coupling compound is as follows:

2. Use according to claim 1, characterized in that, The preparation method of the proline derivative-Rhodamine B coupling compound comprises the following steps: A mixture of proline methyl ester hydrochloride and N-methyl morpholine is dissolved in anhydrous dichloromethane, cooled to 0℃, and then Rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are added in sequence; the obtained solution is stirred at room temperature for amidation reaction; after the reaction is completed, extraction, washing, drying, filtration, vacuum concentration and column chromatography are performed to obtain the proline methyl ester-Rhodamine B coupling compound. The proline methyl ester-Rhodamine B coupling compound is dissolved in a mixed solution of water and THF, and lithium hydroxide is added under ice bath, and then slowly raised to room temperature; after the hydrolysis reaction is completed, acidification is performed to pH = 2, and then extraction, washing, drying, filtration, vacuum concentration and column chromatography are performed to obtain the proline-Rhodamine B coupling compound.

3. Use according to claim 2, characterized in that, The molar ratio of proline methyl ester hydrochloride, N-methyl morpholine, Rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine is 15:20:10:15:1, the reaction temperature is room temperature, and the reaction time is 2-4 hours.

4. Use according to claim 2, characterized in that, The molar ratio of proline methyl ester-Rhodamine B coupling compound and lithium hydroxide is 5:15, the reaction temperature is room temperature, and the reaction time is 1-2 hours.

5. The use according to claim 1, characterized in that, The proline derivative-Rhodamine B coupling compound has the ability of being absorbed by plant root systems and conducted in the aboveground parts of plants. The crop is soybean, corn and tobacco; The structure of the proline derivative-Rhodamine B coupling compound is as follows: The preparation method of the proline derivative-Rhodamine B coupling compound comprises the following steps: A mixture of proline methyl ester hydrochloride and N-methyl morpholine is dissolved in anhydrous dichloromethane, cooled to 0℃, and then Rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are added in sequence; the obtained solution is stirred at room temperature for amidation reaction; after the reaction is completed, extraction, washing, drying, filtration, vacuum concentration and column chromatography are performed to obtain the proline methyl ester-Rhodamine B coupling compound. The proline methyl ester-Rhodamine B coupling compound is dissolved in a mixed solution of water and THF, and lithium hydroxide is added under ice bath, and then slowly raised to room temperature; after the hydrolysis reaction is completed, acidification is performed to pH = 2, and then extraction, washing, drying, filtration, vacuum concentration and column chromatography are performed to obtain the proline-Rhodamine B coupling compound. The molar ratio of proline methyl ester hydrochloride, N-methyl morpholine, Rhodamine B, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine is 15:20:10:15:1, the reaction temperature is room temperature, and the reaction time is 2-4 hours. The molar ratio of proline methyl ester-Rhodamine B coupling compound and lithium hydroxide is 5:15, the reaction temperature is room temperature, and the reaction time is 1-2 hours. The proline derivative-Rhodamine B coupling compound has the ability of being absorbed by plant root systems and conducted in the aboveground parts of plants.

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