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, Ukraine). Before starting to work with Mistletoe Eradicator in your country, please check the environmental regulation,&#160;get permissions for the use of herbicides from the relevant authorities of the country, where Mistletoe Eradicator will be operated, and obtain agreement on its use with the local human communities (farmers, landowners, etc.).
1. Long-term preparations
<ol>
	<li>Briefly introduce yourself or/and other users of Mistletoe Eradicator into the basics of mistletoe biology and ecology, and approaches to its control.</li>
	<li>Optional: Assess the mistletoe incidence, the degree of infestation, host tree mortality rates, and the degree of mistletoe biotic damage of the host trees (e.g., crone defoliation and water stress in host branches acropetal to mistletoe exophyte) to select the priority of mistletoe control on the most infested host trees based on the approaches described in20,30,32,37,38.</li>
	<li>Check weather forecast including wind direction and strength, temperature, probability of rain, snowing, fog, or frost with a reliable meteorological service. Operate Mistletoe Eradicator under dry weather and under adequate light conditions to see the working area clearly.</li>
</ol>
2. Assembly of Mistletoe Eradicator
NOTE: All components of Mistletoe Eradicator are publicly available in supermarkets and gardener stores and are better to be custom-tailored and connected with the help of the professional welding engineer. Details and equipment used for the assembly of Mistletoe Eradicator must be modified and adjusted based on the country of use and availability of the components in an open market.
<ol>
	<li>Prepare all components according to the list and (Figure 1)36 assemble them as follows: acquire a wooden/plastic/metal telescopic or non-telescopic pole with a light telescopic handle, up to 3 m in length, and connect a pruning saw safely and firmly to the pole (Figure 1A; Figure 3A); prepare a container (10-150 mL) with a metal screw plug cap having a custom-drilled 2 mm aperture (inner diameter) for the growth regulator (Figure 1B) and a nozzle with a 2 mm aperture (e.g., stainless steel hollow-through lamp with hole screw). 
	NOTE: The commercial off-the-shelf manual pole saw with further installation of the container for the growth regulator might also be used to construct the Mistletoe Eradicator. Light weight facilitates the work at high peripheral branches of the host tree both from the ground and from the tree. The apertures of both screw plug cap and nozzle might vary in between 2-5 mm and define the herbicide dropping speed. The optimal aperture is 2 mm.</li>
	<li>Firmly connect all metal details with the welding gun in a way that one side of the nozzle (Figure 1B-4) is connected by welding to the metal screw plug cap (Figure 1B-3) tightly fitting the container (Figure 1B-2), while its opposite side is welded to the upper outer corner of the saw blade (Figure 1B-1). Keep a pore (Figure 1B-5) at the point of the attachment of the nozzle and the cutter blade open (Figure 1D).</li>
	<li>Fill the container with water for the dropping test, close the screw plug cap firmly and twist the pole in working position - the sharp surface of the saw blade downwards, the container upwards and upside down. Ensure that the liquid slowly drops down on the saw blade (Figure 2A (inset), Figure 3C).</li>
	<li>In case of clogging, clean the 2 mm-wide nozzle using fine needle (e.g., with a syringe needle or a fine sewing needle) until the clog is removed and the herbicide dropping is restored.</li>
	<li>For transportation or storage, empty the container and secure Mistletoe Eradicator with a saw cover or scabbard. 
	CAUTION: Misuse of Mistletoe Eradicator may result in serious or fatal injuries, so the sharp surface of the blade must be covered.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63455/63455fig01.jpg" /> 
Figure 1: Variants of Mistletoe Eradicator tool. (A-D) An original Indian prototype constructed, tested on walnuts in Kashmir Valley, and patented in India36: (A) the terminal unit of the tool, which needs to be adjusted on the wooden/plastic/metal telescopic or non-telescopic pole with a length up to 3 m; (B) the junction between the pruning saw blade (1) and the container (2) with the drilled metal screw plug cap with 2 mm aperture (3) welded to the nozzle with 2 mm aperture (4) ending with a pore (5); (C) a pore (5) at the point of the joint of the nozzle with the pruning saw blade; (D) the slow dropping of herbicide through the screw plug cap from the container to the nozzle, and from the nozzle to the pruning saw blade through a pore (5); general scheme. (E) and (F) provide details of an alternative version of Mistletoe Eradicator constructed in Ukraine from a commercial tree manual pole saw (s); perforated garden saw with pole handle (p); (b-n)&#160;adjustable bolt-nut connection between the screw cap tube (t) with rounded false bottom and the saw blade. Scale bars for (A) is 5 cm and for (B-D, F) is 2 cm. <a href="https://www.jove.com/files/ftp_upload/63455/63455fig01large.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/63455/63455fig02.jpg" /> 
Figure 2: Mistletoe Eradicator operation mode. (A) from the ground; (B) from the tree by a certified arborist. <a href="https://www.jove.com/files/ftp_upload/63455/63455fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63455/63455fig03.jpg" /> 
Figure 3: A working prototype of Mistletoe Eradicator tested on walnut trees in Kashmir valley in action. (A, B)&#160;A demonstrative tool set up without the growth regulators in hands of the operator. (C)&#160;The pruning position of Mistletoe Eradicator in relation to V. album exophyte. At the hidden side of the tool the slow release of the growth regulator from the container through the pore at the distal end of the nozzle occurs at the site of pruning. (D)&#160;Alternative on-the-tree operation mode and eradication of V. album parasitizing&#160;the peripheral host branch. <a href="https://www.jove.com/files/ftp_upload/63455/63455fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Herbicide preparation
<ol>
	<li>Choose local, commercially available analogue of mistletoe growth regulator(s), ethephon (2-Chloroethylphosphonic acid) or glyphosate (N-(Phosphonomethyl)glycine. Check the correspondence of the chosen herbicides to local legislation since ecological permissions and licenses might be necessary.</li>
	<li>Dilute plant growth regulators according to their specification sheets and manufacturer instructions to prepare stock solutions for short-term storage (not longer than 1-2 days) at 4 &#176;C. On the day of work, prepare 4% -10% working solutions from stock solutions ex tempore. 
	NOTE: Treatments with the growth regulators must be performed by certified workers and arborists according to the current legislation in the country of operation.</li>
</ol>
4. On-site safety rules
<ol>
	<li>For all operator(s) and assistant(s) allowed to operate Mistletoe Eradicator from the ground, provide sufficient personal protection (Figure 2A; Figure 4B): nonslip steel toe work boots; liquid-proof, chemical-resistant coverall or a suit with a hood; a helmet; a chemical respirator due to the risk of inhaling herbicide fumes; an eye/face shield/protecting goggles that conforms to ANSI Z87.1 or CE safety standard; chemical-resistant gloves to protect hands from mechanical damage and/or chemical injuries or heavy nonslip gloves for improved grip. In the neighborhood of the operation area, ensure water access is available in case of emergency.</li>
	<li>Ensure that only licensed arborists with at least one assistant is allowed to operate Mistletoe Eradicator from the tree, using safe gear, bright ropes, arborist lanyards and other professional equipment (Figure 2B). Do not use Mistletoe Eradicator in recreational places. Mark the operation zone and trees with bright stripe tape to prevent the presence of outsiders.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63455/63455fig04.jpg" /> 
Figure 4: An alternative version of Mistletoe Eradicator tested in Ukraine. (A) Free-hand ground-based pruning of the European mistletoe (Viscum album subsp. album L.)&#160;exophyte parasitizing the red buckeye (Aesculus pavia L.) branch in M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Kyiv, Ukraine. (B) An arborist with the tool. (C) Close-up of the tool on the tree bifurcation. (D) Pruned exophyte, (E) cable tie (c) and adjustable bolt-nut connection (b-n)&#160;between the screw cap tube (t) with rounded false bottom and the perforated garden saw blade (s) saw blade with traces of the demonstrative liquid (prepared using a dry E-160&#1089; food color additive) mimicking the growth regulator. (F)&#160;V. album exophyte stub treated with the demonstrative liquid. Scale bar is 2 cm. <a href="https://www.jove.com/files/ftp_upload/63455/63455fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Mistletoe Eradicator operation mode
<ol>
	<li>Prune the mistletoe foliage before their fruiting with sticky viscin-covered berries, and after the harvesting of the host trees. Remove the saw cover or scabbard from the tool and check the reliability of the connection between all parts of Mistletoe Eradicator. Do not operate a damaged, improperly adjusted, or not completely or insecurely assembled tool. 
	CAUTION: Do not hit rocks, stones, or other foreign objects with the blade of Mistletoe Eradicator. Do not cut solid metal, sheet metal, plastics or any non-wood materials with it.</li>
	<li>Evaluate the distance from the operator to the targeted mistletoe on the peripheral host branches and adjust the appropriate length of the telescopic stick to get free access to the attachment point of mistletoe with the host tree (Figure 2A). Ensure that there are no obstructions in the way of Mistletoe Eradicator toward the mistletoe on the tree.</li>
	<li>Find an appropriate saw position (~45&#176; of inclination) in relation to the mistletoe exophyte present in the closest proximity to the host branch/stem (Figure 2A (inset), Figure 3C, Figure 4A).</li>
	<li>Fill the container up to &#190; of its volume with only one growth regulator in working concentration, close the screw plug cap firmly and place Mistletoe Eradicator in a non-working position - the sharp surface of the saw blade upwards, the container downwards, no liquid dropping (Figure 1A).</li>
	<li>Stand closer to the tree and place the tool in the working position by twisting the pole (Figure 2A, inset). Operate Mistletoe Eradicator with both hands while maintaining a safe distance from the assistant(s) and any bystanders (Figure 2A).</li>
	<li>Ensure a good firm grip on the tool and prune an exophyte with short sawing movements of the cutting blade (Figure 4A). Ensure that the distal end of the saw blade does not touch the neighboring host branches and/or stem which might damage them and the container.</li>
	<li>Add more growth regulator as it gets exhausted. The amount of herbicide for one V. album stub varies between 1-5 mL depending on the nozzle diameter.</li>
	<li>After the pruning of one mistletoe exophyte, place the Mistletoe Eradicator in non-working position and let the air come inside the container through the pore. Relocate to another mistletoe exophyte, place Mistletoe Eradicator in working position and repeat the procedure. 
	NOTE: If the Mistletoe Eradicator is operated from the tree by a trained arborist (Figure 2B), the rules for high-altitude work in the country of operation must be followed.</li>
</ol>
6. Mistletoe foliage piling and finishing
<ol>
	<li>Pile the pruned exophytes between the host trees. Mistletoe pruned using Mistletoe Eradicator cannot be collected for pharmacological, ritual, or forage purposes because of the potential danger posed by the growth regulators to human and animal health.</li>
	<li>Dispose the growth regulators following manufacturer instructions and local environmental regulations.</li>
	<li>Clean the tool mechanically after work, empty and thoroughly wash the container with tap water and dry it. 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.</li>
	<li>Protect the sharp part of the Mistletoe Eradicator with a saw cover or scabbard.</li>
</ol>

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<li>León-Bañuelos, L. A., Endara-Agramont, A. R., Gómez-Demetrio, W., Martínez-García, C. G., Nava-Bernal, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Arceuthobium+globosum+using+unmanned+aerial+vehicle+images+in+a+high+mountain+forest+of+central+Mexico%5BTitle%5D)+AND+%22Journal+of+Forest+Research%22%5BJournal%5D)">Identification of Arceuthobium globosum using unmanned aerial vehicle images in a high mountain forest of central Mexico.</a> Journal of Forest Research. 31, 1759-1771 (2020).</li>
<li>Kotan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parasitic+bacteria+and+fungi+on+common+mistletoe+%28Viscum+album+L.%29+and+their+potential+application+in+biocontrol%5BTitle%5D)+AND+%22Journal+of+Phytopathology%22%5BJournal%5D)">Parasitic bacteria and fungi on common mistletoe (Viscum album L.) and their potential application in biocontrol.</a> Journal of Phytopathology. 161, 165-171 (2013).</li>
<li>Szmidla, H., Tkaczyk, M., Plewa, R., Tarwacki, G., Sierota, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+common+mistletoe+%28Viscum+album+L.%29+on+Scots+pine+forests+%E2%88%92+a+call+for+action%5BTitle%5D)+AND+%22Forests%22%5BJournal%5D)">Impact of common mistletoe (Viscum album L.) on Scots pine forests − a call for action.</a> Forests. 10 (847), 1-15 (2019).</li>
<li>Mathiasen, R. L., Nickrent, D. L., Shaw, D. C., Watson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mistletoes%3A+pathology%2C+systematics%2C+ecology%2C+and+management%5BTitle%5D)+AND+%22Plant+Disease%22%5BJournal%5D)">Mistletoes: pathology, systematics, ecology, and management.</a> Plant Disease. 92, 988-1006 (2008).</li>
<li>Mistletoe Management. SAFE TREES. , Hlinky, Brno. Available from: 
 <a target="_blank" href="https://www.safetrees.cz/specialni-ochrana-zelene/index.html">https://www.safetrees.cz/specialni-ochrana-zelene/index.html</a> (2020).</li>
<li>Lichter, J. M., Reid, M. S., Berry, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+methods+for+control+of+leafy+mistletoe+%28Phoradendron+spp.%29+on+landscape+trees%5BTitle%5D)+AND+%22Journal+of+Arboriculture%22%5BJournal%5D)">New methods for control of leafy mistletoe (Phoradendron spp.) on landscape trees.</a> Journal of Arboriculture. 17 (5), 127-130 (1991).</li>
<li>Adams, D. H., Frankel, S. J., Lichter, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+when+using+ethephon+for+suppressing+dwarf+and+leafy+mistletoe+infestations+in+ornamental+landscape%5BTitle%5D)+AND+%22Journal+of+Arboriculture%22%5BJournal%5D)">Considerations when using ethephon for suppressing dwarf and leafy mistletoe infestations in ornamental landscape.</a> Journal of Arboriculture. 19 (6), 351-357 (1993).</li>
<li>Hoyt, H. M., Hornsby, W., Huang, C. H., Jacobs, J. J., Mathiasen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dwarf+mistletoe+control+on+the+Mescalero+Apache+Indian+Reservation%2C+New+Mexico%5BTitle%5D)+AND+%22Journal+of+Forestry%22%5BJournal%5D)">Dwarf mistletoe control on the Mescalero Apache Indian Reservation, New Mexico.</a> Journal of Forestry. 115 (5), 379-384 (2017).</li>
<li>Mistletoe Eradicator. Indian Patent. , Sher-E-Kashmir University of Agricultural Sciences and Technology of Kashmir. 340843 (2017).</li>
<li>SAS Institute Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SAS%2FACCESS+9.4+Interface+to+ADABAS%5BTitle%5D)+AND+%22SAS+Institute+Inc%22%5BJournal%5D)">SAS/ACCESS 9.4 Interface to ADABAS.</a> SAS Institute Inc. , Cary, NC, USA. (2019).</li>
<li>Sananse, S. L., Maidapwad, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+analysis+of+two-way+ANOVA+using+data+transformation+techniques%5BTitle%5D)+AND+%22International+Journal+of+Scientific+Research%22%5BJournal%5D)">On analysis of two-way ANOVA using data transformation techniques.</a> International Journal of Scientific Research. 3 (11), 480-483 (2014).</li>
<li>Kartoolinejad, D., Hosseini, S. M., Akbarinia, M., Shayanmehr, F., Mirnia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relationship+among+infection+intensity+of+Viscum+album+with+some+ecological+parameters+of+host+trees%5BTitle%5D)+AND+%22International+Journal+of+Environmental+Research%22%5BJournal%5D)">The relationship among infection intensity of Viscum album with some ecological parameters of host trees.</a> International Journal of Environmental Research. 1 (2), 143-149 (2007).</li>
<li>Queijeiro-Bolaños, M., González, E. J., Martorell, C., Cano-Santana, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competition+and+facilitation+determine+dwarf+mistletoe+infection+dynamics%5BTitle%5D)+AND+%22Journal+of+Ecology%22%5BJournal%5D)">Competition and facilitation determine dwarf mistletoe infection dynamics.</a> Journal of Ecology. 5 (3), 775-785 (2016).</li>
<li>deBruyn, R. A., Paetkau, M., Ross, M. A., Godfrey, D. V., Friedman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermogenesis-triggered+seed+dispersal+in+dwarf+mistletoe%5BTitle%5D)+AND+%22Nature+communications%22%5BJournal%5D)">Thermogenesis-triggered seed dispersal in dwarf mistletoe.</a> Nature communications. 6, 6262(2015).</li>
<li>Watson, D. M., Rawsthorne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mistletoe+specialist+frugivores%3A+latter-day+%26%2334%3BJohnny+Appleseeds%26%2334%3B+or+self-serving+market+gardeners%5BTitle%5D)+AND+%22Oecologia%22%5BJournal%5D)">Mistletoe specialist frugivores: latter-day &#34;Johnny Appleseeds&#34; or self-serving market gardeners.</a> Oecologia. 172 (4), 925-932 (2013).</li>
<li>Michael, J. L., Talley, K. L., Fishburn, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Forest+herbicide+washoff+from+foliar+applications%5BTitle%5D)+AND+%22Proceedings+on+the+45th+Annual+Meeting+Southern+Weed+Science+Society%22%5BJournal%5D)">Forest herbicide washoff from foliar applications.</a> Proceedings on the 45th Annual Meeting Southern Weed Science Society. , Southern Weed Science Society. Champaign, IL. 236-243 (1992).</li>
<li>Han, S., Berry, A. M., Reid, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+ways+to+control+mistletoe%5BTitle%5D)+AND+%22Proceedings%3A+Fortieth+Annual+California+Weed+Conference%22%5BJournal%5D)">New ways to control mistletoe.</a> Proceedings: Fortieth Annual California Weed Conference. , Sacramento. (1988).</li>
</ol>

SKUAST-Kashmir reserves copyright of the Mistletoe Eradicator tool under patent number 340843 of Indian Patents. The authors declare no other conflict of interests.

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><tbody><tr><td>&lt;strong&gt;Components of Indian Mistletoe Eradicator*&lt;/strong&gt;</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&lt;br/&gt; 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&lt;br/&gt; fLzjNFL</td></tr><tr><td>The pruning saw blade</td><td>Lee Valley</td><td>&amp;nbsp;09A0356</td><td>https://www.leevalley.com/en-us/shop/garden/garden-care/saws/76626-pruning-saw-blade?item=09A0356</td></tr><tr><td>&lt;strong&gt;Components of an alternative Ukrainian Mistletoe Eradicator*&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aluminium Telescopic Hand &lt;em&gt;Gardena Combisystem&lt;/em&gt; 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&amp;amp;keywords&lt;br/&gt; =cable%2Bties&amp;amp;qid=1642924567&amp;amp;sprefix&lt;br/&gt; =Cable%2Btie%2Caps%2C162&amp;amp;sr=8-1-spons&amp;amp;spLa=ZW5jcnlwdGVkUXVh&lt;br/&gt; bGlmaWVyPUEyTU9QMEo2WUp&lt;br/&gt; YMjBLJmVuY3J5cHRlZElkPUEwM&lt;br/&gt; jYzMDkxQUtWODVWU00xU0ZYJ&lt;br/&gt; mVuY3J5cHRlZEFkSWQ9QTA1O&lt;br/&gt; Tc2NjUyVzdHV0E4UUZOWkU1Jn&lt;br/&gt; dpZGdldE5hbWU9c3BfYXRmJmF&lt;br/&gt; jdGlvbj1jbGlja1JlZGlyZWN0JmRv&lt;br/&gt; Tm90TG9nQ2xpY2s9dHJ1ZQ&amp;amp;th=&lt;br/&gt; 1</td></tr><tr><td>Garden saw &lt;em&gt;GARTNER 540/360 mm&lt;/em&gt; with bar handle</td><td>G&amp;auml;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 &lt;em&gt;INTERTOOL HT-3111&lt;/em&gt;</td><td>ROZETKA</td><td>Code&amp;nbsp;&amp;nbsp;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&amp;amp;_sid=a8a2c15c9&amp;amp;_ss=r&amp;amp;&lt;br/&gt; 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>&lt;strong&gt;Chemicals&lt;/strong&gt;</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 (&amp;ldquo;Promoter&amp;rdquo;)</td><td>Fil, India Ltd</td><td>n/a</td><td>https://www.filindustries.com/our-businesses.php</td></tr><tr><td>&lt;strong&gt;Protection*&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gloves &lt;em&gt;Tegera Cut Control&lt;/em&gt;</td><td>EJENDALS</td><td>TEGERA 9121</td><td>https://www.ejendals.com/globalassets/inriver/resources/9121_productsheet_en.&lt;br/&gt; pdf</td></tr><tr><td>Helmet &lt;em&gt;CAMP Rockstar&lt;/em&gt;</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 &lt;em&gt;Stihl&lt;/em&gt;</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...

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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 ...

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3.9K Views.  Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir. Maretak zijn hotspots voor biodiversiteit, evenals parasieten die water en voedingsstoffen opnemen uit hun gastheerbomen. De raketverdelger die in dit protocol wordt getoond, helpt om de maretak die zich op de bomen verspreidt efficiënt te beheersen. Deze technieken maken het mogelijk om mechanische en chemische maretakcontrolemethoden te combineren, door gelijktijdig mechanisch maretak te snoeien en de groeiregulator gericht in de stomp te laten vallen.Voordat u met de maretak-eradic...

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

A Venturi Effect Can Help Cure Our Trees

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e. pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called &#34;trunk injection&#34; or &#34;trunk infusion&#34; (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels&#39; cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.

Compared to the more traditional hole-based methods, most of which require the tree to be drilled, tools with lenticular blades transform the basics of endotherapy easing the closure of the wound and allowing the natural uptake of the solutions.

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. ...

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Biochemistry

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Biology

Temporary Translocation of Entire Mistletoe Plants to Understand the Mechanistic Basis of Animal Foraging Decisions

Fruiting mistletoes present a model system for understanding decisions made by foraging animals when locating food. Where, when, and how animals find food is central to many ecological questions, relating to the basis of individual foraging decisions and the extent to which these decisions are innate or acquired. Ecologists have paid particular attention to frugivores, quantifying their preference for fruits with specific shapes, colors, or scents, which, over evolutionary time, confer selection for suites of traits in their favored plants whose seeds they disperse.
This work outlines a novel experimental approach to manipulating food plant occurrence and measuring the response of wild, free-living animals, ideally suited to studying the evolutionary origin and ecological maintenance of seed dispersal. This &#34;cut and paste&#34; protocol involves removing an entire fruiting mistletoe plant from its host and either returning it to its original location or moving it to a novel location, affixing it to a &#39;pseudo-host&#39; of the same or different tree species. By counting visits to the mistletoe and noting the duration, species, and behaviors, a series of comparisons can discern the most important factors affecting foraging decisions and the consequences for both plant and animal. Here, the protocol is illustrated with a case study to determine between-guild differences in mistletoe frugivory.
The experimental approach teases apart the mechanistic basis of search image formation and refinement, spatial learning, interspecific differences in foraging strategies, and how these changes modify seed dispersal effectiveness. Finally, potential modifications are considered with respect to addressing other questions on foraging ecology, plant-animal interactions, and coevolution.

This work outlines a simple experimental procedure to quantify behavioral drivers of foraging decisions in free-living animals, temporarily relocating mistletoe plants to novel locations and measuring visitation rates.

Fruiting mistletoes present a model system for understanding decisions made by foraging animals when locating food. Where, when, and how animals find food ...

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

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