Name: mistletoe eradicator - a novel tool for simultaneous mechanical and chemical control of mistletoe
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The authors thank plant taxonomist Dr. Norbert Holstein (Nees-Institute for the Biodiversity of Plants, Bonn University, Germany) for his kind help in the primary identification of mistletoe species. Permissions from the following farmers and owners of walnut plantations in Kashmir, who offered their infected trees for our experiments, were obtained, namely from Abdul Rashid Reshi (R/O Village Reshii Mohalla, Khag, Budgam), Habib-Ullah Sheikh (R/O village Trapii, Khag, Budgam), Showket Ahmad Rasray (R/O Village Hamchii Pora, Budgam), Ghulam Hassan Shah (R/O Nagbal, Khag, Budgam), and Javeed Ahmad Qureshi (R/O village Lowahi Pora, Khag, Budgam).The drawings of Mistletoe Eradicator operation mode was made by the fine artist Natalia Pendiur (Kyiv, Ukraine) and are highly appreciated. Maksym Lashchenko (Olomouc, Czech Republic) kindly helped with the image processing and technical descriptions.

The growth regulators used in the work are available in the market after approval by the Central Insecticide Board (http://ppqs.gov.in/central-insecticides-board). Moreover, personal invitations and permissions of the farmers and owners of walnut plantations, who offered their infected trees for experiments, were obtained. Experiments presented here were done by a certified arborist in the closed research and monitoring zone of M.M. Gryshko National Botanical Garden of National Academy of Sciences of Ukraine (Kyiv, Ukrai...

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Mistletoes cannot be treated exclusively as pests, since many of them play a crucial role in the ecosystem as biodiversity hotspots and connecting tropical and topical hubs for multiple organisms, behaving more like epiphytes than parasites with negligible negative consequences for their hosts6,8. Therefore, the measures aimed to control their spread have to be highly selective and based on the understanding of both biology and biotic associations of definite mis...

Mistletoes are a taxonomically diverse group of aerial stem hemiparasites belonging to the Loranthaceae, Viscaceae, Santalaceae, Amphorogynaceae, and Misodendraceae families (order Santalales). They attach to the susceptible host trees by a specific anatomical bridge called a haustorium (endophyte) and are largely dependent on the host for water and nutrient supply, but are still able to photosynthesize via the green aerial portion called exophyte1,2. Although mistletoes may be considered hotspots of diversity providing food and shelter for various organisms, some mistletoe species are indeed biological pirates, affecting host health, fitness, and lifespan by the reducing their annual apical and lateral growth, flowering and fruiting as well as facilitating attacks of biological invaders such as fungi and bacteria3,4,5. At the same time, mistletoes are intensely used in both traditional and folk medicine, are components of compost, mulch, forage, and act as seasonal ecotourism attractions6,7,8.
Some of the notorious parasitic mistletoes are the dwarf mistletoes (Arceuthobium spp.; Santalaceae) reducing the growth of coniferous trees in North America9&#160;and Mexico10; Tapinanthus (Blume) Rchb. mistletoes (Loranthaceae) attacking cacao (Theobroma cacao L.) and Citrus L. plantations in Africa11,12; hairy mistletoes (Taxillus tomentosus (Heyne ex Roth) van Tiegh.; Loranthaceae) affecting the Indian Euphorbiaceae trees, Phyllanthus emblica L. and Phyllanthus indofischeri Bennet, both known locally as Amla13; and the European mistletoe (Viscum album subsp. album L.; Viscaceae) having a broad host spectrum (more than 450 tree species)14,15 and rapidly spreading in Europe with its range shifted northwards16.
Walnut (Juglans regia L.) is an important horticultural cash crop in Kashmir valley constituting approximately 85% of India&#39;s annual walnut production17. Besides other biotic stresses such as leaf blotch, anthracnose, crown galls, stem cankers, walnut blight, or lace bug (Paracopium cingalense (Walker, 1873); Heteroptera: Tingidae) that attack the walnuts in Kashmir Valley, the V. album, locally called banada, kaw-khoor or nalkachul, also heavily infests these trees and can also rarely parasitize poplars (Populus L.; poplars in Kashmir valley are&#160;secondary hosts)18. Such a localized infestation might be explained by the possible concomitant introduction of V. album at the early developmental stages of 2-3 years, when its exophytes are almost invisible, with the planting material of walnuts from some plant nurseries. In turn, local poplar trees, the widespread susceptible primary host of this mistletoe in Europe, might be attended by birds vectoring sticky viscin-covered mistletoe fruits to the neighboring J. regia orchards.
Besides walnuts, leafy mistletoes parasitize other nut-producing trees, for example, almonds (Prunus dulcis (Mill.) D.A. Webb) are attacked by V. album in the Mediterranean Europe and in Crimea (Ukraine) as well as by the representatives of Phoradendron Nutt. genus in North America14,15,19; common hazel (Corylus avellane L.) by V. album in Europe14,15; pecan (Carya illinoinensis (Wangenh.) K.Koch) by oak mistletoe (Phoradendron flavescens (Pursh) Nutt.) in the southeastern United States20,21, and V. album in Europe14; macadamia (Macadamia F. Muell.) by shiny-leaved mistletoe (Benthamina alyxifolia (F.Muell. ex Benth.) Tiegh.), concomitant coast mistletoe (Mullerina celastroides (Sieb. ex. Schult &#38; Schult. f.) Tiegh.), erect mistletoe (Amyema congener (Sieber ex Schult. &#38; Schult. f.) Tiegh.), smooth mistletoe (Dendrophthoe glabrescens (Blakeley) Barlow), and long-flowered mistletoe (Dendrophthoe vitelline (Sieb. ex. Schult &#38; Schult.f.) Tiegh.) in Australia22 (Watson, 2018); kolanut (Cola nitida (Vent.) Schott &#38; Endl.) by Phragmanthera incana (Schumach.) Balle and Tapinanthus bangwensis (Engl. &#38; K.Krause) Danser in Nigeria23; and cashew (Anacardium occidentale L.) by Psittacanthus plagiophyllus Eichler in Amazonian savanna24.
Currently, the strategy of mistletoe control develops in frames of the integrated pest management paradigm combining mechanical, chemical, and biological means to reduce infestations4,7,25,26,27,28. An environmentally friendly mistletoe biocontrol method involves the use of the natural enemies of mistletoe such as bacteria, necrotrophic fungi, herbivores, or even epiparasitic mistletoes3,4,7,29,30 but have limited applications due to the non-specificity and small-scale of action of the biotic agents. Although cutting and pruning are recognized as the most traditional and efficient means of mistletoe control26,30,31,32, exophytes frequently resprout. For spraying of mistletoe exophytes with chemicals, mistletoe stubs or host stems are injected with systemic herbicides such as glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB), or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)3,33 or ethylene-releasing defoliants and ripening enhancers such as ethephon or (2-chloroethyl)-phosphonic acid)34,35. The combination of exophyte pruning to 2-3 cm stubs along with ethephon&#160;application to cut end of mistletoe stubs with a fine brush resulted in reduced V. album resprouting. This inspired the development of Mistletoe Eradicator, which can perform mechanical and chemical control of mistletoe growth simultaneously.
Here, we introduce an easy-to-handle and broadly available tool called Mistletoe Eradicator, which combines the benefits of mechanical and chemical means of mistletoe control and prevents its resprouting. The tool allows the certified arborists, plant pathologists, foresters, horticulturists, silviculturists, plant ecologists, experienced gardeners and other instructed users to deal with mistletoe challenge from the ground, from aerial platforms or from the trees. The tool performs a targeted pruning of mistletoe exophyte with the simultaneous dropping of a small amount of growth regulator directly on the pruned mistletoe stem. Mistletoe Eradicator was patented in India36 and used for the successful control of an increasing V. album subsp. album infestation on walnuts in Kashmir Valley. An updated version of this tool was designed in Ukraine and introduced to local arborists to practice in field tests with just a food color additive to track the herbicide dropping. It is noteworthy that Mistletoe Eradicator can be easily assembled and used for the control of other host-aggressive mistletoe species at the sites of their infestation in accordance with the national environmental law and after the approval of the local human communities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components of Indian Mistletoe Eradicator*</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bosch 2607010538 HSS-G Drill Bit Set, Silver</td>
			<td>Bosch</td>
			<td>2607010538</td>
			<td>https://www.amazon.co.uk/Bosch-Professional-2607010538-HSS-G-Silver/dp/B001IBMPL2</td>
		</tr>
		<tr>
			<td>Container- Aluminium Bottle- with screw plug Cap</td>
			<td>Suparna Exports, Pvt. LTD</td>
			<td>n/a</td>
			<td>http://www.aluminumbottlecans.com/aluminum-bottles-with-screw-plugs.php</td>
		</tr>
		<tr>
			<td>Drill GSR12V-300HXB22</td>
			<td>Bosch</td>
			<td>GSR12V-300HXB22</td>
			<td>https://www.boschtools.com/us/en/boschtools-ocs/cordless-screwdrivers-gsr12v-300hxb22-207019-p/</td>
		</tr>
		<tr>
			<td>Genuine C04AL-1487425 engine valve guide for engine spare parts</td>
			<td>Shangchai</td>
			<td>C04AL-1487425</td>
			<td>https://www.alibaba.com/product-detail/Genuine-C04AL-1487425-engine-valve-guide_60704258097.html?spm=a2700.details.0.0.2cfb6b8b2k 
			JBOB</td>
		</tr>
		<tr>
			<td>HBCHEN Handheld Welder Gun Portable Welding Machine, 220V Small Welding Machine and 110V Arc Welder</td>
			<td>HBCHEN</td>
			<td>YB320</td>
			<td>https://www.amazon.com/HBCHEN-Handheld-Portable-Eliminate-Experience/dp/B097BWBCMB</td>
		</tr>
		<tr>
			<td>The nozzle: stainless steel outer bolt hollow-through lamp with hole screw</td>
			<td>n/a</td>
			<td>M6M8M10M12</td>
			<td>https://www.aliexpress.com/item/32950601820.html?spm=a2g0o.seo_rank.0.0.62642d1 
			fLzjNFL</td>
		</tr>
		<tr>
			<td>The pruning saw blade</td>
			<td>Lee Valley</td>
			<td>&#160;09A0356</td>
			<td>https://www.leevalley.com/en-us/shop/garden/garden-care/saws/76626-pruning-saw-blade?item=09A0356</td>
		</tr>
		<tr>
			<td>Components of an alternative Ukrainian Mistletoe Eradicator*</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium Telescopic Hand Gardena Combisystem 160-290 cm</td>
			<td>ROZETKA</td>
			<td>Code 03720-20</td>
			<td>https://rozetka.com.ua/202484293/p202484293/characteristics/</td>
		</tr>
		<tr>
			<td>Amazon Basics Multi-Purpose Cable Ties - 4-Inch/100mm</td>
			<td>Amazon Basics</td>
			<td>n/a</td>
			<td>https://www.amazon.com/AmazonBasics-Multi-Purpose-Cable-Ties-200-Piece/dp/B087MKMSDY/ref=sr_1_1_sspa?crid=3FEZFXGOR4VPC&#38;keywords 
			=cable%2Bties&#38;qid=1642924567&#38;sprefix 
			=Cable%2Btie%2Caps%2C162&#38;sr=8-1-spons&#38;spLa=ZW5jcnlwdGVkUXVh 
			bGlmaWVyPUEyTU9QMEo2WUp 
			YMjBLJmVuY3J5cHRlZElkPUEwM 
			jYzMDkxQUtWODVWU00xU0ZYJ 
			mVuY3J5cHRlZEFkSWQ9QTA1O 
			Tc2NjUyVzdHV0E4UUZOWkU1Jn 
			dpZGdldE5hbWU9c3BfYXRmJmF 
			jdGlvbj1jbGlja1JlZGlyZWN0JmRv 
			Tm90TG9nQ2xpY2s9dHJ1ZQ&#38;th= 
			1</td>
		</tr>
		<tr>
			<td>Garden saw GARTNER 540/360 mm with bar handle</td>
			<td>G&#228;rtner</td>
			<td>Code 80001003</td>
			<td>https://gartner-tools.com/ru/product/pila-sadovaja-540-360-mm-s-rukojatkoj-pod-shtangu-gartner/</td>
		</tr>
		<tr>
			<td>Loppers INTERTOOL HT-3111</td>
			<td>ROZETKA</td>
			<td>Code&#160;&#160;263723496</td>
			<td>https://rozetka.com.ua/intertool_ht_3111/p263723496/characteristics/</td>
		</tr>
		<tr>
			<td>M4 / 4mm DOOR HANDLE SCREWS BOLTS 20-22 mm</td>
			<td>Boltworld</td>
			<td>n/a</td>
			<td>https://boltworld.co.uk/collections/m4-bolts/products/m4-4mm-door-handle-screws-bolts-9mm-50mm-lengths-cupboard-door-drawer-knob?variant=15820185469021</td>
		</tr>
		<tr>
			<td>M4 Hexagon Weld Nut A4 Stainless Nut DIN 929</td>
			<td>Boltworld</td>
			<td>n/a</td>
			<td>https://boltworld.co.uk/products/m4-hexagon-weld-nut-a4-stainless-nut-din-929?_pos=1&#38;_sid=a8a2c15c9&#38;_ss=r&#38; 
			variant=32145946706013</td>
		</tr>
		<tr>
			<td>Sarstedt 5ml Screw Cap Tube with Rounded False Bottom and Cap Assembled, with Print, 92x15.3, PP</td>
			<td>Sarstedt</td>
			<td>n/a</td>
			<td>https://www.amazon.com/Sarstedt-Rounded-Bottom-Assembled-92x15-3/dp/B07GXZ51BN</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium salt of Glyphosate 71% SG EXCEL MERA 71</td>
			<td>Excel Crop Care</td>
			<td>n/a</td>
			<td>http://www.excelcropcare.com/product/domestic/crop-protection/herbicides/excel-mera-71.php</td>
		</tr>
		<tr>
			<td>E-160c*</td>
			<td>EUROIMPEX</td>
			<td>n/a</td>
			<td>https://euroimpex.net.ua/ru/krasiteli/</td>
		</tr>
		<tr>
			<td>Ethephon (&#8220;Promoter&#8221;)</td>
			<td>Fil, India Ltd</td>
			<td>n/a</td>
			<td>https://www.filindustries.com/our-businesses.php</td>
		</tr>
		<tr>
			<td>Protection*</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves Tegera Cut Control</td>
			<td>EJENDALS</td>
			<td>TEGERA 9121</td>
			<td>https://www.ejendals.com/globalassets/inriver/resources/9121_productsheet_en. 
			pdf</td>
		</tr>
		<tr>
			<td>Helmet CAMP Rockstar</td>
			<td>CAMP</td>
			<td>ID: 0202</td>
			<td>https://www.camp-usa.com/outdoor/product/helmets/rock-star-helmet/</td>
		</tr>
		<tr>
			<td>Mesh visor Stihl</td>
			<td>STIHL</td>
			<td>n/a</td>
			<td>https://www.stihl.co.uk/STIHL-Products/Personal-protective-equipment/Face-and-ear-protection/22184-1545/Face-hearing-protection-nylon-mesh.aspx</td>
		</tr>
		<tr>
			<td>SAS/STAT</td>
			<td>SAS Institute Inc. 2013, USA</td>
			<td></td>
			<td>statistical analysis software</td>
		</tr>
		<tr>
			<td>*materials and equipment marked with asterisks are changeable and can be modified and adjusted basing on the country of the use and avaliability of the components in the market. Moreover, Mistletoe Eradicator can be custom-tailored by the professional welding engineer.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mistletoe eradicator - a novel tool for simultaneous mechanical and chemical control of mistletoe

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic and topic associations with various organisms and at the same time acting as biological pirates, exhausting the host trees by stealing water and nutrients via the haustorium. Many crop and timber trees are attacked by mistletoes, for example, dwarf mistletoes parasitize conifers, representatives of genus Tapinanthus target guava, and Phoradendron mistletoe target pecans. Recently, an increasing infestation of the European mistletoe (Viscum album subsp. album L.) in monoculture stands of common walnuts (Juglans regia L.)&#160;was recorded for the first time during 2018 in different regions of Kashmir Valley, India. The common chemical approach based on the spraying of mistletoe exophytes with the growth regulators, ethephon and glyphosate, was not efficient since V. album resprouted in the next vegetation season. Hand pruning of mistletoe was time-consuming, and a polyethylene covering resulted in the localized rotting of host tissues. The most efficient approach to eradicate mistletoe was the use of a novel tool called Mistletoe Eradicator, which allows to use mechanical and chemical means of mistletoe control simultaneously. It consists of a manipulating pole with the terminal pruning saw having a container filled with the growth regulator, from which the liquid slowly drops onto the saw blade through the nozzle. It allows working both from the ground and in the tree canopy. After the targeted application of 5%-10% ethephon or glyphosate to the base of the just pruned V. album stems (stubs), exophyte resprouting was significantly reduced 9 months after the pruning as compared to the only pruned or sprayed exophytes. An alternative variant of Mistletoe Eradicator was constructed in Ukraine filled just with a food color additive to track the liquid dropping and was successfully tested.

Different V. album management approaches were tested in Kashmir (India) in walnut plantations during 2012-2016 before the use of the Mistletoe Eradicator, such as targeted foliar spraying with glyphosate and ethephon in different concentrations, pruning/clipping of mistletoe exophytes, secure covering of the wound with polyethylene wrap and application of the same herbicides to the cut ends of 2-3 cm-long mistletoe stubs.
Each approach was tested on five V. album exophytes on...

Watch this Scientific Journal Video about Mistletoe Eradicator - A Novel Tool for Simultaneous Mechanical and Chemical Control of Mistletoe at JoVE.com

Mistletoe Eradicator - A Novel Tool for Simultaneous Mechanical and Chemical Control of Mistletoe

The protocol describes an easy-to-handle tool called Mistletoe Eradicator which combines mechanical and chemical approaches and is efficient in the control of mistletoe infestation on the host trees. The manuscript uses the European mistletoe (Viscum album subsp. album L.) as an example.

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic ...

Mistletoes are biodiversity hotspots, as well as parasites that uptake water and nutrients from their host trees. The missile eradicator shown in this protocol, helps to efficiently control the mistletoe spread on the trees. These techniques allows the combining of mechanical and chemical mistletoe control methods, by simultaneous mechanical mistletoe pruning, and targeted dropping of the growth regulator into the stub.Before the working with the mistletoe eradicator, please check the local environmental regulations, get the permissions for herbicide's use, agree with the local community, and get a basic understanding of mistletoe biology. Before handling the eradicator, ensure that the personal protective equipment complies with the safety rules, and dress on. Before operation, remove the saw cover from the tool, and check the reliability of the connection between all parts of the mistletoe eradicator.To assemble the mistletoe eradicator, fill the container with the color additive water for the dropping test, to track the flow of the liquid in this protocol. Close the screw plug cap firmly, and twist the pole in the working position, with the sharp surface of the saw blade facing downwards, and the container inverted. ensure that the liquid drops down on the saw blade.In case of clogging, clean the two millimeter wide nozzle using a fine needle until the clog is removed, and the dropping is restored. Then prepare working solutions by diluting the stock solutions of the plant growth regulators. Prune the mistletoe exophyte foliage before buds development, and after the harvesting of the host trees.Adjust the length of the telescopic stick to get free access to the attachment point of the mistletoe on the branches, or at the of the stem host tree. Ensure that there are no obstructions in the way of the mistletoe eradicator toward the mistletoe. Next, fill 3/4 of the container with one growth regulator solution, close the screw plug cap firmly, and place the mistletoe eradicator in a non-working position, with the sharp surface of the saw blade upwards, the container downwards, and no liquid dropping.Stand closer to the tree and place the tool in the working position by twisting the pole. Position the saw at approximately 45 degrees of inclination, in relation to the mistletoe exophyte present in the closest proximity to the host branch. Operate the mistletoe eradicator with both hands, while maintaining a safe distance from others.Ensure a firm grip on the tool, and prune and exophyte with short sawing movements of the cutting blade. Ensure that the distal end of the saw blade does not touch the neighboring host branches or stem. After pruning, place the mistletoe eradicator in non-working position, and let the air come inside the container through the pore.Relocate to another exophyte mistletoe, place mistletoe eradicator in working position, and repeat the procedure. After pruning, pile the pruned exophytes between the host trees. Clean the eradicator mechanically, empty and thoroughly wash the container with tap water, and dry it.Protect the sharp part of the mistletoe eradicator with a saw cover. Discard the disposable protective gloves and respirators in special containers. Thoroughly rinse the reusable cloth, boots, helmets, and eye protection with tap water.Dry and store separately. The mistletoe eradicator was tested for the treatment of walnut trees infested with Viscom album in the cashmere valley. Exophytes were pruned using the mistletoe eradicator loaded with ethephon or glyphosate as growth regulators.While distilled water was used as mock control. After nine months of observation, the 2 to 4 centimeter long stubs, treated with 10%ethephon, showed 5%resprouting. And the stubs treated with 10%glyphosate, showed resprouting of 6%While stubs exposed to water as mock control, showed 97%resprouting.An alternative version of mistletoe eradicator was designed in Ukraine. Pruning of the European mistletoe exophytes, parasitizing the red buckeye tree, was done using a colored solution, mimicking the growth regulator. Demonstrating the simultaneous mechanical and chemical control of mistletoe growth.The mistletoe eradicator reduces the regrowth of mistletoes after their simultaneous pruning and chemical treatment. And therefore it is a useful tool in the integrated strategy of mistletoe management. This tool can be used for the control of pathogenic leafy mistletoes, such as the European Viscum album, American Phoradendron, Australian Amyema, and others.

mistletoe-eradicator-novel-tool-for-simultaneous-mechanical-chemical

Research

JoVE Journal

Environment

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...