The research was supported by the National Research Council (CNR) of Italy. The authors thank GIRE members for collecting seed samples and are grateful to Alison Garside for revising the English.

1. Seed Sampling and Storage
<ol>
	<li>Monitor cultivated fields for unjustified poor herbicide performance, i.e., not due to adverse climatic conditions or low quality herbicide treatments.</li>
	<li>Collect a seed sample from one species at a time and assign a unique code. Mature seeds are usually collected before crop harvesting from plants that had survived the herbicide treatment(s). Timely monitor to observe if the seeds are shed by mother plant when mature.</li>
	<li>Fill in a form for each sample indicating the assigned unique code, name of species, collection date, GPS coordinates, municipality, farmer&#8217;s name, field size, infestation level, crop, herbicide(s) used during the season and historical records of the field.</li>
	<li>Collect seeds from at least 30 randomly selected plants that are representative of the field infestation. Ensure that the seed sample contains at least 5,000 mature seeds. For an obligate outcrossing weed species (e.g., Lolium spp. or Amaranthus spp.), reduce the number of plants to 10-15, keeping the total number of seeds around 5,000 11.</li>
	<li>Sub-sample the field if patches of weeds are scattered over large areas (more than a hectare) as different herbicide resistant biotypes are selected.</li>
	<li>Store seeds in unsealed paper bags labeled with the unique code assigned.</li>
	<li>Allow moisture to evaporate but do not expose seeds to high temperature (i.e., avoid leaving them in a car under the sun) or to extreme temperature fluctuations to avoid induction of secondary dormancy.</li>
	<li>Clean (remove chaff, de-hull seeds, etc.) and store them at ambient temperature in a dry room. After conducting the first resistance tests, store the seeds for long periods of time in a dark room at 4 &#176;C, preferably in vacuum-sealed plastic bags. In this way seeds preserve their viability for a significantly longer time.</li>
</ol>
2. Seed Dormancy Breaking
NOTE: Seed dormancy provides a flexible and efficient mechanism that enables weeds to adapt and persist in agro-ecosystems. To break dormancy and allow seed germination, different protocols have to be used depending on weed species, i.e., the type of dormancy 16. 
There are three main ways to remove dormancy:
<ol>
	<li>Vernalization 
	NOTE: To obtain simultaneous germination and seedling emergence, a period of seed vernalization ranging from a few days to a week is required to remove physiological dormancy from many species: e.g., Amaranthus retroflexus, Chenopodium album, Lolium spp., Avena fatua, Polygonum persicaria, Phalaris paradoxa17-19. A longer period of up to 15 days is required for Papaver rhoeas, Cyperus difformis and Ammania coccinea and up to 30 days for Schoenoplectus mucronatus20.
	<ol>
		<li>Put some deionized water in plastic dishes. Cut two layers of filter paper and soak them in the water, remove any excess. Place the air-dried seeds on the paper. Transfer the plastic dishes to a fridge at 4 &#176;C for the required period of time.</li>
	</ol>
	</li>
	<li>Scarification 
	NOTE: Some weed species are more recalcitrant to germination than others due to mechanical dormancy, i.e. seed coat characteristics, and necessitate the use of a chemical scarification using sulfuric acid to germinate 21.
	<ol>
		<li>Prepare a beaker with concentrated sulfuric acid (95-98%). Prepare a beaker full of water. Put the seeds in an envelope of non-woven fabric.</li>
		<li>Soak e.g., Echinochloa spp. or Sorghum halepense seeds for 20 min or 5 min, respectively, in concentrated sulfuric acid.</li>
		<li>Take the envelope out of the beaker using a pair of tweezers and put it in the beaker full of water. Open the envelope, put the seeds in a small colander and rinse them thoroughly under running water.</li>
	</ol>
	Variant: A. plantago-aquatica needs a different protocol 22, 23
	<ol>
		<li>Soak the seeds for 2 min in chloroform. Rinse the seeds with deionized water and dry them with absorbent paper. Dunk the seeds in 80% sulfuric acid for 5 min.</li>
		<li>Put the seeds in a small colander and rinse them thoroughly under running water.</li>
	</ol>
	</li>
	<li>Post-harvest seed maturation 
	NOTE: Seeds of other weed species do not germinate at all for a few months after maturity, regardless of the method used to break dormancy.
	<ol>
		<li>Store the seeds for a period of at least 3-4 months at RT and low humidity and then follow the above protocols for dormancy breaking (e.g., Oryza sativa var. sylvatica or P. rhoeas).</li>
	</ol>
	</li>
</ol>
3. Seed Germination
<ol>
	<li>Place seeds to be germinated in plastic dishes containing 0.6% (m/v) agar with 0.1% potassium nitrate (KNO3) added:
	<ol>
		<li>Prepare a solution of agar at 0.6% + 0.1% KNO3 using deionized water. Dissolve the agar in a microwave oven.</li>
		<li>Pour the agar solution into plastic dishes. Cool the substrate and then put in the seeds.</li>
	</ol>
	Variant: Soil can also be used as a substrate in plastic dishes. This practice is particularly efficient for Echinochloa spp., S. halepense and Lolium spp.</li>
	<li>Place plastic dishes in a germination cabinet for about a week with light and temperature conditions depending on the optimum conditions for each weed species. For most winter species, the temperature range is 15/25 &#176;C night/day and 12 hr photoperiod with neon tubes providing a Photosynthetic Photon Flux Density (PPFD) of 15-30 &#181;mol m-2 sec-1. For many summer species, the temperature range is 15/30 &#176;C night/day. 
	Variant: Some species, such as S. halepense, need a heat treatment. Therefore after the scarification, seeds of S. halepense are subjected to the following conditions: cycles of 4 hr at 45 &#176;C and 20 hr at 24 &#176;C for three days in the germination cabinet, and then three days in normal conditions.</li>
</ol>
4. Seedling Transplanting and Growth
<ol>
	<li>Transplant fifteen to twenty seedlings into plastic trays (325 x 265 x 95 mm) filled with a standard potting mix (60% silty loam soil, 15% sand, 15% agriperlite and 10% peat &#8212; by volume). 
	NOTE: Transplanting, instead of direct sowing, allows a uniform stand of plants at the same growth stage to be obtained, which is an important pre-condition to optimize the performance of the herbicide treatment.</li>
	<li>Identify each tray with a barcode including all information for the unique identification: population code, herbicide being tested, replicate number and progressive tray number.</li>
	<li>Place trays in a heated greenhouse and water plants as needed to maintain the substrate at or near field capacity. 
	NOTE: The growth temperature varies depending on the weed species. Often tests are done during autumn/winter/spring, so light is supplemented using 400 W metal-halide lamps, which provide a PPFD of about 150 &#181;mol m-2 sec-1 and a 12 hr photoperiod 24, 19. Summer weed species with C4 photosynthetic cycle usually require higher light intensity and therefore tests are done in late spring-summer or the supplemented light intensity is about 400 &#181;mol m-2 sec-1 with a 14 hr photoperiod.</li>
	<li>Use a different protocol for some weed species infesting paddy rice, e.g., A. plantago-aquatica, S. mucronatus and C. difformis as described in 22.
	<ol>
		<li>Transplant the seedlings into polystyrene trays with 24 round cells (55 mm diameter, 64 mm deep) filled with 60% silty loam soil, 30% sand and 10% peat (by volume).</li>
		<li>Set the trays in 12 cm deep plastic containers filled with water and battened down by screwed stainless steel rods to prevent them floating (Figure 1).</li>
		<li>Maintain water level in the containers at 1-2 cm below the level of the soil surface and add 1.5 g of copper sulfate to each container (which contains 10-12 L of water) to avoid the proliferation of algae.</li>
	</ol>
	</li>
</ol>
5. Herbicide Treatments
<ol>
	<li>Treatments with pre-emergence herbicides:
	<ol>
		<li>After about three days in the germination cabinet as described in section 3, transplant the germinating seeds into plastic trays containing the substrate described above and cover with a layer of soil (about 1 cm). This is a critical step in order to ensure that seedlings will not emerge due to the herbicide effect rather than an excessive burial depth.</li>
		<li>Take the substrate to field capacity by placing the trays, which have a few holes at the bottom, on saucers filled with water.</li>
		<li>One day after transplanting, treat the trays with the pre-emergence herbicide 25.</li>
		<li>Keep the substrate at or near field capacity by adding water as needed both from above and below by capillarity from the saucer. This procedure favors the permanence of the herbicide at the proper depth (i.e., where the germinating seeds are) for a good treatment efficacy.</li>
	</ol>
	</li>
	<li>Treatments with post-emergence herbicides:
	<ol>
		<li>Spray plants when they reach the 2-3 leaf stage (i.e., growth stage 12-13 of the Extended BBCH growth scale 26).</li>
		<li>Starting from the day after the treatment, set the irrigation system according to the water requirements of the weed species and the season (e.g., for Echinochloa spp. it delivers water for 3 min 4 times per day, at regular intervals from 9 am to 9 pm). Water is distributed using an automatic sprinkler irrigation system. 
		Variant: glyphosate is applied at plant stage BBCH 14&#8211;21.</li>
	</ol>
	</li>
	<li>Herbicide preparation and distribution. 
	NOTE: All herbicides (pre- and post-emergence) are applied as commercial formulations with recommended surfactants at two doses, recommended field dose (1x) and three times that (3x).
	<ol>
		<li>If needed, prepare the surfactant solution in bulk according to the label instructions; the final concentration is usually expressed as a percentage of the final volume (e.g., 0.3%) or as volume to be distributed per unit area (e.g., 1 L ha-1).</li>
		<li>Use the surfactant solution as solvent for the herbicide (solute) solution in order to keep the right concentration of the active ingredient. Prepare the most concentrated herbicide solution first (3x). Calculate the quantity of commercial product to be dissolved in the surfactant solution (or in deionized water if a surfactant is not necessary) using the following equation: 
		Doseherb = [(Dosefield x Dosemax) x Vfin] / Vdel 
		Where: Doseherb = herbicide dose (ml), Dosefield = herbicide field dose (ml ha-1), Dosemax = maximum dose delivered, Vfin = final volume of the solution (L), Vdel = volume delivered by the bench sprayer (L ha-1).</li>
		<li>Dilute (2:1, v/v) the herbicide solution 3x to prepare the less concentrated one (1x). This procedure reduces the chance of making mistakes when weighing or pipetting the herbicides. Herbicide solution concentration is expressed as volume to be distributed per unit area (L ha-1).</li>
		<li>Start the sequence of treatment with the lower herbicide dose (1x). In this way there is no need to wash the spraying cabinet between two treatments with the same herbicide.</li>
		<li>Distribute the herbicide solution using a precision bench sprayer delivering 300 L ha-1 (&#177;1%), at a pressure of 215 kPa, and a speed of 0.75 m sec-1, with a boom equipped with three flat-fan (extended range) hydraulic nozzles.</li>
		<li>Wash the spraying cabinet twice when the herbicide is changed using bleach 1% (v/v) and then rinse. 
		Variant: glyphosate is applied with a spray volume of 200 L ha-1 27. 
		NOTE: Particular attention has to be paid when highly biological herbicides, such the sulfonylureas sulfometuron or flazasulfuron, are used. In the latter case do one washing with a bleach solution and another with ammonia (2.5% v/v), followed by a careful rinse with water.</li>
	</ol>
	</li>
</ol>
6. Collection and Analysis of the Data
<ol>
	<li>Through a barcode reader, which automatically identifies each tray, record the number of plants that survived the treatment as well as the Visual Estimated Biomass (VEB). Plants are assessed as being dead if they show no active growth regardless of color or other appearance.
	<ol>
		<li>Make the assessment three or four weeks after treatment (WAT) depending on the herbicides tested (e.g., three WAT for ACCase inhibitors and four WAT for ALS inhibitors or glyphosate).</li>
		<li>Evaluate the general treatment efficacy by including a susceptible population (check S) in all experiments, i.e., a population collected in a site which was never or seldom treated with herbicides.</li>
		<li>Express plant survival as percentage of the number of treated plants, counted just before the herbicide treatment, and calculate the standard error (S.E.) per mean value (mean value of the two replicates).</li>
		<li>The VEB is obtained through a visual comparison of plant biomass between treated and not-treated check of the same population 25, 28. A score, ranging from 10 for plants not affected by the herbicide (compared with the not-treated check) to 0 when the plants are clearly dead, is given to each treated tray.</li>
	</ol>
	</li>
	<li>Ascribe populations to four categories based on the results obtained from treatments with two herbicide doses: S when less than 5% of plants survived the herbicide dose 1x, SR when survivors ranged from 5% to 20% at herbicide dose 1x, R when more than 20% of plants survived the herbicide dose 1x and RR when survivors are more than 20% at herbicide dose 1x and more than 10% at herbicide dose 3x 17.</li>
</ol>

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The authors declare that they have no competing financial interests.

Several steps within the protocols are critical for a successful assessment of herbicide resistance in a population: 1) seeds should be collected when mature from plants that had survived the herbicide treatment(s). Maturation of the seeds on the mother plant is crucial to avoid difficulties in seed germination later; 2) the proper storage of seeds is recommended to avoid proliferation of molds that would prevent germination; 3) seedlings should be treated at the right growth stage, as reported on the label of the herbic...

Herbicides are the most extensively used weed control measure, accounting for up to 50% of the global plant protection market 1. They are relatively cheap tools, avoid labor-intensive and time-consuming soil cultivation practices, and ultimately result in cost-effective, safe and profitable food production 2. However, the great phenological and genetic variability present in many weed species, together with an over-reliance on herbicide use, frequently results in the selection of herbicide-resistant weed populations. The introduction of selective herbicides with a very specific metabolic target 3-5 has dramatically increased the number of resistance cases over the years. To date, 240 weed species (140 dicots and 100 monocots) worldwide have evolved resistance to different herbicide Sites of Action (SoA) 4. This is a major concern for weed management and more in general for sustainable crop production.
Early detection of resistance, based on reliable tests, frequently performed in a greenhouse, is a key step to manage herbicide resistant weeds. Different approaches have been developed according to the aims, required level of accuracy, time and resources available, as well as the weed species considered 6-12. However, when confirmation of the resistance status of a new weed biotype is required (i.e., a group of individuals that share several physiological characteristics, including the ability to survive one or more herbicides belonging to a particular group used at a dose that would normally control them), a robust whole plant bioassay needs to be performed in a controlled environment 4, 11.
A biotype is seldom resistant to just one herbicide. Each biotype is therefore characterized by a certain resistance pattern, i.e., number and type of SoA of the herbicides it is resistant to, and by a given resistance level to each herbicide 13. The early and reliable determination of the pattern of cross or multiple resistance 5, 14 is important for field resistance management.
It is worth mentioning that herbicide resistance has nothing to do with the natural tolerance that some weed species exhibit towards some herbicides, e.g., dicot species vs. ACCase-inhibiting herbicides, monocot species vs. 2,4-D, Equisetum arvense vs. glyphosate.
This paper presents a robust approach for testing putative herbicide resistant biotypes sampled in fields where poor control by herbicide(s) had been reported. Relevant variants to the standard protocols in relation to the weed species involved are presented. The advantages over alternative techniques/protocols based on either whole plant bioassays using only one herbicide dose 15,or treating seeds in Petri dishes 8 are related to the higher reliability and the possibility of inferring the resistance level because of the inclusion of two herbicide doses in the experiments. However, for routine resistance testing, the same methods can be applied at only one herbicide dose, so reducing the costs.
As well as allowing confirmation of the resistance status, the information obtained can be used for both optimizing the following research steps and/or devising sound resistance management strategies.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Paper bags</td>
			<td>Celcar SAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic dishes</td>
			<td>ISI plast S.p.A.</td>
			<td>SO600</td>
			<td>Transparent plastic</td>
		</tr>
		<tr>
			<td>Sulfuric acid 95-98%</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven fabric</td>
			<td>Carretta Tessitura</td>
			<td>Art.TNT17</td>
			<td>Weight&#160; 17 gr m-&#178;</td>
		</tr>
		<tr>
			<td>Chloroform &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium nitrate &#62;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>P8394</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic containers</td>
			<td>Giganplast</td>
			<td>1875/M</td>
			<td>600 x 400 x 110 mm</td>
		</tr>
		<tr>
			<td>Plastic trays</td>
			<td>Piber plast</td>
			<td>G1210A</td>
			<td>325 x 265 x 95 mm</td>
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		<tr>
			<td>Polystyrene trays</td>
			<td>Plastisavio</td>
			<td>S24</td>
			<td>537x328x72 mm, 24 round cells (6x4)&#160;</td>
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		<tr>
			<td>Copper sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>451657</td>
			<td></td>
		</tr>
		<tr>
			<td>Agriperlite</td>
			<td>Blu Agroingross sas</td>
			<td>AGRI100</td>
			<td></td>
		</tr>
		<tr>
			<td>Peat</td>
			<td>Blu Agroingross sas</td>
			<td>TORBA250</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Germination cabinet</td>
			<td>KW</td>
			<td>W87R</td>
			<td></td>
		</tr>
		<tr>
			<td>Nozzles</td>
			<td>Teejet</td>
			<td>&#160;XR11002-VK, TP11001-VH</td>
			<td>The second type of nozzles are used only for glyphosate</td>
		</tr>
		<tr>
			<td>Barcode generator</td>
			<td>Toshiba TEC</td>
			<td>SX4</td>
			<td></td>
		</tr>
		<tr>
			<td>Labels with barcode</td>
			<td>Felga</td>
			<td>TT20200</td>
			<td>Stick-in labels with rounded corners</td>
		</tr>
		<tr>
			<td>Barcode reader</td>
			<td>Cipherlab</td>
			<td>8300-L</td>
			<td>Portable data terminal</td>
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		<tr>
			<td>Bench sprayer</td>
			<td>-</td>
			<td>-</td>
			<td>Built in house</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td colspan="2">HERBICIDES INCLUDED IN THE RESULTS:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial product</td>
			<td>Active ingredient</td>
			<td>Company</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Altorex</td>
			<td>imazamox</td>
			<td>BASF</td>
			<td></td>
		</tr>
		<tr>
			<td>Azimut&#160;</td>
			<td>florasulam</td>
			<td>Dow AgroSciences</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopower</td>
			<td></td>
			<td>Bayer Crop Science</td>
			<td>Surfact to be used with Hussar WG</td>
		</tr>
		<tr>
			<td>Dash</td>
			<td></td>
			<td>BASF</td>
			<td>Surfact to be used with Altorex</td>
		</tr>
		<tr>
			<td>Granstar&#160;</td>
			<td>tribenuron-methyl</td>
			<td>Dupont</td>
			<td></td>
		</tr>
		<tr>
			<td>Gulliver&#160;</td>
			<td>azimsulfuron</td>
			<td>Dupont</td>
			<td></td>
		</tr>
		<tr>
			<td>Hussar WG&#160;</td>
			<td>iodosulfuron</td>
			<td>Bayer Crop Science</td>
			<td></td>
		</tr>
		<tr>
			<td>Nominee&#160;</td>
			<td>bispyribac-Na</td>
			<td>Bayer Crop Science</td>
			<td></td>
		</tr>
		<tr>
			<td>Roundup</td>
			<td>glyphosate</td>
			<td>Monsanto</td>
			<td></td>
		</tr>
		<tr>
			<td>Trend</td>
			<td></td>
			<td>Dupont</td>
			<td>Surfact to be used with Granstar and Gulliver</td>
		</tr>
		<tr>
			<td>Viper&#160;</td>
			<td>penoxsulam</td>
			<td>Dow AgroSciences</td>
			<td></td>
		</tr>
		<tr>
			<td>Weedone LV4</td>
			<td>2,4-D</td>
			<td>Isagro</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To assess the resistance status of a putative resistant population, it is fundamental to include a susceptible check in the assay in order to verify the herbicide efficacy. The results of a screening test conducted on P. rhoeas populations, a weed infesting wheat fields, are reported in Figure 2, where the efficacy of four post-emergence herbicides on a susceptible check (09-36) and on the suspected resistant one (10-91) are presented. Population 09-36 was completely controlled by the ALS inhibi...

Watch this Scientific Journal Video about Protocols for Robust Herbicide Resistance Testing in Different Weed Species at JoVE.com

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

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 ...

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14.4K Views.  National Research Council (CNR), Italy. 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.

Video: Protocols for Robust Herbicide Resistance Testing in Different Weed Species

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

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 ...

Potentiodynamic Corrosion Testing

Different metallic materials have different polarization characteristics as dictated by the open circuit potential, breakdown potential, and passivation potential of the material. The detection of these electrochemical parameters identifies the corrosion factors of a material. A reliable and well-functioning corrosion system is required to achieve this.
Corrosion of the samples was achieved via a potentiodynamic polarization technique employing a three-electrode configuration, consisting of reference, counter, and working electrodes. Prior to commencement a baseline potential is obtained. Following the stabilization of the corrosion potential (Ecorr), the applied potential is ramped at a slow rate in the positive direction relative to the reference electrode. The working electrode was a stainless steel screw. The reference electrode was a standard Ag/AgCl. The counter electrode used was a platinum mesh. Having a reliable and well-functioning in vitro corrosion system to test biomaterials provides an in-expensive technique that allows for the systematic characterization of the material by determining the breakdown potential, to further understand the material's response to corrosion. The goal of the protocol is to set up and run an in vitro potentiodynamic corrosion system to analyze pitting corrosion for small metallic medical devices.

Here, we present a protocol to set up and run an in vitro potentiodynamic corrosion system to analyze pitting corrosion for small metallic medical devices.

Different metallic materials have different polarization characteristics as dictated by the open circuit potential, breakdown potential, and passivation ...

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 ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides in attempts to control its populations eliminates natural enemies and competitor flower thrips species, thereby increasing its populations. An unsustainable situation develops with concomitant resistant pest populations, secondary pest outbreaks, and environmental degradation. Integrated pest management utilizes knowledge of pest and natural enemy relationships to implement tactics that are environmentally friendly and sustainable. Minute pirate bugs are the most important worldwide predators of thrips. They can suppress and ultimately control Frankliniella species flower thrips. Flower samples taken at least weekly are needed to understand predator-prey dynamics. Shown here is the sampling of the flowers of fruiting vegetables and companion plants to estimate the densities of individual thrips and minute pirate bug species. Representative data illustrates how the protocol is used to determine the efficacy of management tactics over time and how to evaluate the benefits of predation by minute pirate bugs. The sampling protocol is similarly adaptable to sampling thrips and minute pirate bugs in other plant species hosts.

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides ...