This work was supported by the National Natural Science Foundation of China (No. 42277039).

1. Germination of wheat seeds
<ol>
	<li>Select 1,000 wheat seeds (Jimai 20) with complete granules, intact embryos, and uniform size (length: 6 mm &#177; 0.5 mm).</li>
	<li>Transfer 333.3 mL of 30% H2O2 solution to a 1 L volumetric flask and dilute with deionized water to prepare 1 L of 10% H2O2 solution. Immerse the wheat seeds in 10% H2O2 solution for 15 min to disinfect the seed surface (Figure 1).</li>
	<li>Rinse the wheat seeds 5x with running sterile water for 10 s each time.</li>
	<li>Spread the wheat seeds evenly with the embryos pointing up in a glass Petri dish containing moist sterile filter paper (Figure 2). Place the Petri dish in an artificial climate incubator at 30 &#176;C and 80% relative humidity. Culture the wheat seeds in the dark for 3 days until they germinate and take root.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64741/64741fig01.jpg" /> 
Figure 1: Disinfection of the wheat seeds. The wheat seeds were soaked in 10% H2O2 solution (in a beaker) for 15 min to disinfect the seed surface. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64741/64741fig02.jpg" /> 
Figure 2: Germinating the wheat seeds. The wheat seeds were spread evenly in a glass Petri dish containing moist sterile filter paper. The Petri dish was placed in an artificial climate incubator to germinate the wheat seeds. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cultivation of wheat seedlings
<ol>
	<li>Dissolve 551 mg of Hoagland&#39;s basal salt mixture in 1 L of deionized water to prepare 1/2 Hoagland nutrient solution (containing 0.75 mmol/L K2SO4, 0.1 mmol/L KCl, 0.6 mmol/L MgSO4, 4.0 &#215; 10&#8722;2 mmol/L FeEDTA, 1.0 &#215; 10&#8722;3 mmol/L H3BO3, 1.0 &#215; 10&#8722;3 mmol/L MnSO4, 1.0 &#215; 10&#8722;3 mmol/L ZnSO4, 1.0 &#215; 10&#8722;4 mmol/L CuSO4, and 5.0 &#215; 10&#8722;6 mmol/L Na2&#160;MoO4).</li>
	<li>After the wheat seeds (step 1.4) have germinated, place 15 wheat seedlings in hydroponic equipment (see Table of Materials) containing 100 mL of 1/2 Hoagland nutrient solution for hydroponics (Figure 3). Place the entire hydroponic apparatus in an artificial climate incubator (see Table of Materials) and incubate for 7 days at 25 &#176;C and 80% relative humidity with a 16 h light/8 h dark photoperiod.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64741/64741fig03.jpg" /> 
Figure 3: Hydroponic cultivation of the wheat seedlings. The wheat seedlings were hydroponically cultivated for 0 days, 3 days, and 7 days in 100 mL of 1/2 Hoagland nutrient solution. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Experiment exposing the wheat plants to imidacloprid solution
<ol>
	<li>After a 7 day hydroponic period, transplant the wheat plants into 1/2 Hoagland nutrient solution containing 0.5 mg/L or 5 mg/L imidacloprid to conduct the imidacloprid exposure experiments. Grow 15 wheat plants in each hydroponic device. Set up 15 hydroponic devices for each imidacloprid concentration group to ensure that adequate samples are taken during sampling.</li>
	<li>Place the entire hydroponic equipment in an artificial climate incubator for 3 days at 25 &#176;C and 80% relative humidity with a 16 h light/8 h dark photoperiod.</li>
	<li>Throughout the exposure period, collect wheat roots (0.2 g per wheat plant) and leaves (0.5 g per wheat plant) daily. Integrate the wheat samples from every fifth hydroponic device as a parallel group and determine the imidacloprid content of the samples.</li>
</ol>
4. Procedure for extracting imidacloprid from wheat
<ol>
	<li>Extraction of imidacloprid from wheat roots
	<ol>
		<li>To avoid experimental errors, wash the wheat roots 4x with running sterile water for 10 s each time to remove any imidacloprid adsorbed on the root surface.</li>
		<li>Shred the wheat roots into approximately 1 cm pieces with scissors (Figure 4). Weigh 10.00 g of the shredded wheat roots and place in a 50 mL centrifuge tube.</li>
		<li>Add 10 mL of acetonitrile to the centrifuge tube and vortex the tube on a vortexer for 1 min. Then, add 4 g of anhydrous MgSO4 and 1.5 g of NaCl to the centrifuge tube and vortex the tube immediately for 30 s. Centrifuge the tube for 5 min at 6,000 x g.</li>
		<li>Aspirate the supernatant with a disposable syringe and pass it through a syringe filter (0.22 &#181;m pore size) to obtain the sample.</li>
	</ol>
	</li>
	<li>Extraction of imidacloprid from wheat leaves (Figure 5)
	<ol>
		<li>Shred the fresh wheat leaves into approximately 1 cm pieces with scissors (Figure 4). Weigh 10.00 g of the shredded wheat leaves and place in a 50 mL centrifuge tube.</li>
		<li>Add 10 mL acetonitrile to the centrifuge tube and vortex the tube on a vortexer for 1 min.</li>
		<li>Add 4 g of anhydrous MgSO4 and 1.5 g of NaCl to the centrifuge tube and vortex the tube immediately for 30 s.</li>
		<li>Centrifuge the tube for 5 min at 6,000 x g.</li>
		<li>After centrifugation, add 2 mL of the supernatant to a 5 mL centrifuge tube containing 50 mg of graphitized carbon black (GCB) and 150 mg of anhydrous MgSO4 (to remove pigment and moisture from the sample), and vortex the centrifuge tube for 30 s (Figure 6). Centrifuge the tube for 5 min at 6,000 x g.</li>
		<li>Aspirate the supernatant with a disposable syringe and pass it through a syringe filter (0.22 &#181;m pore size) to obtain the sample.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64741/64741fig04.jpg" /> 
Figure 4: Shredded wheat roots and leaves. Fresh wheat roots and leaves were shredded using scissors into approximately 1 cm pieces. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64741/64741fig05.jpg" /> 
Figure 5: Extraction of imidacloprid in the wheat leaves. Imidacloprid in the samples was extracted using the QuEChERS method (steps 4.2.1-4.2.4 of the protocol). <a href="https://www.jove.com/files/ftp_upload/64741/64741fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64741/64741fig06.jpg" /> 
Figure 6: Purification of imidacloprid in the wheat leaves. The decontaminant was 50 mg GCB + 150 mg MgSO4. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Quantification of imidacloprid
<ol>
	<li>Quantify the imidacloprid in the sample using liquid chromatography-tandem mass spectrometry (LC-MS-MS),based on a standard curve (y = 696.61x + 56.411, R=1) obtained from 0.2-250 &#181;g/L imidacloprid concentrations. (Figure 7). The mass spectrometer was equipped with a C18 column (100 mm x 2.1 mm, 3 &#181;m) and an electrospray ionization source (ESI+). The elution program and ion source parameters are shown in Table 1.</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64741/64741fig07.jpg" /> 
Figure 7: Chromatogram and mass spectrogram of imidacloprid in the wheat leaves. The upper panel shows a chromatogram of imidacloprid (retention time = 0.93 min). The lower panel shows the mass spectrogram of imidacloprid at 0.93 min, showing the response intensity of the production (m/z = 208.8) of imidacloprid. <a href="https://www.jove.com/files/ftp_upload/64741/64741fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Column temperature</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>Solvent A</td>
			<td>99.9% water/0.1% formic acid (v/v)</td>
		</tr>
		<tr>
			<td>Solvent B</td>
			<td>acetonitrile</td>
		</tr>
		<tr>
			<td rowspan="5">Elution program</td>
			<td>0&#8211;0.5 min, A = 20%</td>
		</tr>
		<tr>
			<td>0.5&#8211;2 min, A = 20%&#8211;50%</td>
		</tr>
		<tr>
			<td>2&#8211;3 min, A = 50%</td>
		</tr>
		<tr>
			<td>3&#8211;3.1 min, A = 50%&#8211;20%</td>
		</tr>
		<tr>
			<td>3.1&#8211;5 min, A=20%</td>
		</tr>
		<tr>
			<td>Flow rate (mL/min)</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>Injection volume (&#956;L)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Capillary temperature (&#176;C)</td>
			<td>330</td>
		</tr>
		<tr>
			<td>Vaporizer temperature (&#176;C)</td>
			<td>350</td>
		</tr>
		<tr>
			<td>Sheath gas flow rate (Arb)</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Aux gas flow rate (Arb)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Spray voltage (V)</td>
			<td>3900</td>
		</tr>
		<tr>
			<td>Collision gas pressure (mTorr)</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>Precursor ion</td>
			<td>256.1</td>
		</tr>
		<tr>
			<td>Product ion/Collision energy (eV)</td>
			<td>208.8/16</td>
		</tr>
	</tbody>
</table>
Table 1: Elution program and ion source parameters of the liquid chromatography-mass spectrometry method.

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The authors declare that they have no conflicts of interest. All the authors have read and approved the manuscript. This work has not been published previously, nor is it being considered by any other peer-reviewed journal.

In recent years, methods for the pretreatment and detection of residues of the pesticide imidacloprid have been frequently reported. Badawy et al.23 used high-performance liquid chromatography to determine the content of imidacloprid in tomato fruit grown under greenhouse conditions and reported good linearity for imidacloprid in the range 0.0125-0.15 &#181;g/mL. Zhai et al.24 used LC-MS-MS to study the residue of imidacloprid in Chinese chives. In the present study, the Qu...

In present day agronomy, the use of pesticides is essential to increase crop yield. Neonicotinoid insecticides alter the membrane potential balance by controlling nicotinic acetylcholine receptors in the insect nervous system, thereby inhibiting the normal conduction of the insect central nervous system, leading to the paralysis and death of the insects1. Compared with traditional insecticides, neonicotinoids have advantages such as novel modes of action, high insecticidal activity, and strong root absorption, making them highly successful in the pesticide market2,3. The sales volume of neonicotinoids was reported to account for 27% of the world pesticide market in 2014. The average annual growth rate of neonicotinoids was 11.4% from 2005 to 2010, of which about 7% was registered in China4,5,6. From the end of 2016 to the first half of 2017, the sales of pesticides in China began to rebound after falling, and the prices of pesticides continued to rise, among which neonicotinoid insecticides showed a significant price increase7. So far, three generations of neonicotinoid insecticides have been developed, each containing pyridine chloride, thiazolyl, and tetrahydrofuran groups of nicotine, respectively8.
Imidacloprid represents the first generation of neonicotinoid insecticides, whose molecular formula is C9H10ClN5O2, and is a colorless crystal. Imidacloprid is used mainly to control pests, such as aphids, planthoppers, mealworms, and thrips9 and can be applied to crops such as rice, wheat, corn, cotton, and vegetables such as potatoes, as well as fruit trees. Due to the long-term, substantial, and continual application of pesticides, both beneficial insects and the natural enemies of pests have been rapidly reduced, and some agricultural pests have become resistant to pesticides, resulting in a vicious circle of applying continual and increasing amounts of pesticides10. In addition, the extensive application of pesticides has led to the deterioration of soil quality, persistent pesticide residues in agricultural products, and other ecological problems, which not only cause significant damage to the agricultural ecological environment11 but also pose a serious threat to human health12. Pesticide spraying severely impacts the growth and quality of soil microbes and soil animals13. The unreasonable or excessive use of pesticides has caused significant security risks to the soil and water environment, animals and plants, and even human life14. In recent years, the problem of excessive pesticide residues in crops has become more severe with the extensive application of pesticides. When imidacloprid was used to increase vegetable yield, the absorption rate of imidacloprid in the vegetables increased with the increase in the amount and residue of imidacloprid15. As a major food crop, both the production and safety of wheat are critical. Therefore, the residue and distribution policies of pesticides used for wheat need to be clarified.
In recent years, many methods have been developed to extract imidacloprid residues from water, soil, and plants. The QuEChERS method (quick, easy, cheap, effective, rugged, and safe) is a new method that combines solid-phase microextraction technology and dispersed solid-phase extraction technology and involves the use of acetonitrile as the extraction solvent and the removal of mixed impurities and water in the sample using NaCl and anhydrous MgSO4, respectively16. The QuEChERS method requires minimal glassware and has simple experimental steps, making it one of the most popular pesticide extraction methods17. For the detection of imidacloprid, a detection limit as low as 1 &#215; 10&#8722;9 g18 has been achieved with liquid chromatography (LC), and 1 &#215; 10&#8722;11 g19 has been achieved with gas chromatography (GC). Due to their high resolution and sensitivity, LC-MS and GC-MS have shown even lower imidacloprid detection limits of 1 &#215; 10-13 to 1 &#215; 10-14 g20,21; these techniques are, therefore, well suited for the analysis of trace imidacloprid residues.
In the present study, imidacloprid was chosen as the target pollutant, and wheat was selected as the test crop to study the distribution of imidacloprid residues in wheat. This protocol details a method for the comprehensive analysis of the enrichment and transfer of the pesticide imidacloprid in wheat by exploring the absorption and storage of imidacloprid in different parts of wheat plants grown under hydroponic conditions. The present study aims to provide a theoretical basis for the risk assessment of pesticide residues in wheat, guide the rational application of pesticides in agricultural production activities to reduce pesticide residues, and improve the safety of crop production.

<table><tbody><tr><td>Acetonitrile</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>01-06-1995</td><td>Suitable for HPLC, gradient grade, &gt;99.9%</td></tr><tr><td>Analytical balance</td><td>Sartorius Lab Instruments Co.Ltd.</td><td>GL124-1SCN</td><td></td></tr><tr><td>Artificial climate incubator&amp;nbsp;&amp;nbsp;</td><td>Shanghai Badian Instrument Equipment Co. Ltd.</td><td>HK320</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf China Co. Ltd.</td><td>Centrifuge5804</td><td></td></tr><tr><td>Disposable syringe</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>Z116866</td><td>Capacity 5 mL, graduated 0.2 mL, non-sterile</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>Y0001970</td><td>European pharmacopoeia reference standard</td></tr><tr><td>Graphitized carbon black (GCB)</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>V900058</td><td>45 &amp;mu;m</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td><td>31642</td><td>30% (w/w)</td></tr><tr><td>Hoagland&amp;rsquo;s Basal Salt Mixture</td><td>Shanghai Yu Bo Biotech Co. Ltd.</td><td>NS1011</td><td>Anhydrous, reagent grade</td></tr><tr><td>Hydroponic equipment</td><td>Jiangsu Rongcheng Agricultural Science and Technology Development Co.Ltd.</td><td>SDZ04BD</td><td></td></tr><tr><td>Hypersil BDS C18 column</td><td>Thermo Fisher Scientific (China) Co. Ltd.</td><td>28103-102130</td><td></td></tr><tr><td>Imidacloprid</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>Y0002028</td><td>European pharmacopoeia reference standard</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich (Shanghai) Trading Co. Ltd.</td><td>208094</td><td>Anhydrous, reagent grade, &gt;97%</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td><td>S9888</td><td>Reagent grade, 99%</td></tr><tr><td>pH meter</td><td>Shanghai Thunder Magnetic Instrument Factory</td><td>PHSJ-3F</td><td></td></tr><tr><td>Phytotron box</td><td>Harbin Donglian Electronic Technology Co. Ltd.</td><td>HPG-280B</td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf China Co. Ltd.</td><td>Research plus</td><td></td></tr><tr><td>Syringe filter</td><td>Sigma-Aldrich (Shanghai) Trading Co.Ltd.</td><td>SLGV033N</td><td>Nylon, 0.22 &amp;micro;m pore size, 33 mm, non-sterile</td></tr><tr><td>Ultra performance liquid chromatography tandem triple quadrupole mass spectrometry</td><td>Thermo Fisher Scientific (China) Co. Ltd.</td><td>UltiMate 3000</td><td></td></tr><tr><td></td><td></td><td>TSQ Quantum Access MAX</td><td></td></tr><tr><td>Vortex mixer</td><td>Shanghai Yetuo Technology Co. Ltd.</td><td>Vortex-2</td><td></td></tr><tr><td>Wheat seed</td><td>LuKe seed industry</td><td></td><td>Jimai 20</td></tr></tbody></table>

determination of the absorption, translocation, and distribution of imidacloprid in wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

The instrument limit of detection (LOD) of imidacloprid was 5.76 &#215; 10&#8722;14 g, and the method&#39;s LOD of imidacloprid in the wheat root or leaf was 0.01 &#181;g/kg; no matrix effect was observed. The recovery yields of imidacloprid in wheat are shown in Table 2. The recovery yields of imidacloprid from the wheat roots exposed to imidacloprid concentrations of 0.5 mg/L and 5 mg/L were 94.0%-97.6% and 98.8%-99.2%, respectively; the coefficients of variation were 1.92% and 0.20%, respec...

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Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

determination-absorption-translocation-distribution-imidacloprid

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1.7K Views.  Shandong Agricultural University. Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Video: Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Biochemistry

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is imperative to investigate the accumulation and translocation of PFAAs within plants. Long-distance transport is an important pathway for PFAAs transferred from the plant leaves to the edible tissues through the phloem. However, it was previously difficult to assess the translocation potential of organic contamination in a short-term exposure period. The split-root experiment provides a solution to effectively uncover the long-distance translocation of PFAAs using a hydroponic experiment, which, in this study, was carried out in two 50 mL centrifuge tubes (A and B), of which centrifuge tube A had 50 mL of one-quarter strength Hoagland sterile nutrient solution, while centrifuge tube B had the same amount of nutrient concentration, and the target PFAAs (perfluorooctane sulfonic acid, PFOS, and perfluorooctane acid, PFOA) added at a given concentration. A whole wheat root was manually separated into two parts and inserted carefully into tubes A and B. The concentration of PFAAs in the roots, shoots of wheat, and solutions in tubes A and B were evaluated using LC-MS/MS, respectively, after being cultured in an incubator for 7 days and harvested. The results suggested that PFOA and PFOS experience a similar long-distance transport process through the phloem from the shoot to the root and could be released into the ambient environment. Thus, the split-root technique can be used to evaluate the long-distance transport of different chemicals.

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is ...

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...

Characterization of Cell Wall-Related Enzymes in RWA-Infested Wheat Cultivars

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as &#946;-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and &#946;-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 &#176;C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. &#946;-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using &#946;-1,3-glucan and &#946;-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 &#176;C, and thermal stability between 30 &#176;C and 70 &#176;C. &#946;-1,3-glucanase substrate specificity was determined at 25 &#176;C and 40 &#176;C using curdlan and barley &#946;-1,3-1,4-glucan substrates. Additionally, the &#946;-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and &#946;-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Author Spotlight: Integrating Biochemical Functions of &#946;-Glucanases and Peroxidase Enzymes in Wheat-RWA Interaction

The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly &#946;-1,3-glucanase and peroxidase, in wheat plants. Their activity levels increase during wheat-RWA interaction and are involved in the plant defense response through cell wall reinforcement, which deters aphid feeding.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

Molecule Delivery in Plants Using a Direct Infusion Device

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Summary

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Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

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Direct Infusion Device for Molecule Delivery in Plants