Name: methodology to test control agents and insecticides against the coffee berry borer hypothenemus hampei
Uploaded: 2022-03-23T14:30:00
Description: Watch this Scientific Journal Video about Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei at JoVE.com

The authors express their thanks to the National Federation of Coffee Growers of Colombia, the assistants of the Department of Entomology (Diana Marcela Giraldo, Gloria Patricia Naranjo), Experiment Station Naranjal, and Jhon F&#233;lix Trejos.

NOTE: This protocol addresses different methods to identify different effects on the CBB under laboratory conditions.
1. Fruit collection
<ol>
	<li>Pick GFs with a developmental age of ~120-150 days after flowering from trees in a coffee plantation early in the morning.</li>
</ol>
2. Fruit disinfection20
<ol>
	<li>Bring around 300 GFs to the laboratory. Select uniformly sized and healthy GFs and withdraw the ...

<ol>
	<li>Mendesil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemicals+used+in+host+location+by+the+coffee+berry+borer,+Hypothenemus+hampei.">Semiochemicals used in host location by the coffee berry borer, Hypothenemus hampei.</a> Journal of Chemical Ecology. 35 (8), 944-950 (2009).</li><li>Jaramillo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee+berry+borer+joins+bark+beetles+in+coffee+klatch.">Coffee berry borer joins bark beetles in coffee klatch.</a> PLoS ONE. 8 (9), 74277 (2013).</li><li>Giraldo-Jaramillo, M., Garcia, A. G., Parra, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+thermal+requirements,+and+estimation+of+the+number+of+generations+of+Hypothenemus+hampei+(Ferrari,+1867)+(Coleoptera:+Curculionidae)+in+the+state+of+S&#227;o+Paulo,+Brazil.">Biology, thermal requirements, and estimation of the number of generations of Hypothenemus hampei (Ferrari, 1867) (Coleoptera: Curculionidae) in the state of S&#227;o Paulo, Brazil.</a> Journal of Economic Entomology. 111 (5), 2192-2200 (2018).</li><li>Benavides, P., G&#243;ngora, C., Bustillo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPM+Program+to+Control+Coffee+Berry+Borer+Hypothenemus+hampei,+with+Emphasis+on+Highly+Pathogenic+Mixed+Strains+of+Beauveria+bassiana,+to+Overcome+Insecticide+Resistance+in+Colombia.">IPM Program to Control Coffee Berry Borer Hypothenemus hampei, with Emphasis on Highly Pathogenic Mixed Strains of Beauveria bassiana, to Overcome Insecticide Resistance in Colombia.</a> IntechOpen. , (2012).</li><li>Mart&#237;nez, C. P., Echeverri, C., Florez, J. C., Gaitan, A. L., G&#243;ngora, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+production+of+two+chitinolytic+proteins+with+an+inhibiting+effect+on+the+insect+coffee+berry+borer,+Hypothenemus+hampei+(Ferrari)+(Coleoptera:+Curculionidae)+and+the+fungus+Hemileia+vastatrix+the+most+limiting+pests+of+coffee+crops.">In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops.</a> AMB Express. 2, 1-11 (2012).</li><li>Padilla, B. E., Acu&#241;a, Z., Vel&#225;squez, C. S., Rubio, G. J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+[alpha]-amylases+from+the+coffee+berry+borer+Hypothenemus+hampei+in+different+plant+species.">Inhibitors of [alpha]-amylases from the coffee berry borer Hypothenemus hampei in different plant species.</a> Revista Colombiana de Entomolog&#237;a. 32 (2), 125-130 (2006).</li><li>Alvarez, J. H., Cortina, H. A., Villegas, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+evaluate+antibiosis+to+Hypothenemus+hampei+Ferrari+in+coffee+under+controlled+conditions.">Methods to evaluate antibiosis to Hypothenemus hampei Ferrari in coffee under controlled conditions.</a> Cenicaf&#233;. 52 (3), 205-214 (2001).</li><li>. New Product in the Integrated Management of the Coffee Berry Borer in Colombia. National Coffee Research Center (Cenicaf&#233;) Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/handle/10778/477">https://biblioteca.cenicafe.org/handle/10778/477</a> (2014)</li><li>Jaramillo, J., Montoya, E., Benavides, P., G&#243;ngora, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beauveria+bassiana+and+Metarhizium+anisopliae+for+the+control+of+coffee+brocade+in+fruits+on+the+ground.">Beauveria bassiana and Metarhizium anisopliae for the control of coffee brocade in fruits on the ground.</a> Revista Colombiana de Entomolog&#237;a. 41, 95-104 (2015).</li><li>Bastidas, A., Vel&#225;squez, E., Benavides, P., Bustillo, A., Orozco, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+preformulated+Beauveria+bassiana+(B&#225;lsam)+Vuillemin,+for+the+control+of+the+coffee+berry+borer.">Evaluation of preformulated Beauveria bassiana (B&#225;lsam) Vuillemin, for the control of the coffee berry borer.</a> Agronomia. 17, 44-61 (2009).</li><li>Villalba-Gault, D., Bustillo, A., Chaves Cordoba, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+insecticides+for+the+control+of+the+coffee+berry+borer+in+Colombia.">Evaluation of insecticides for the control of the coffee berry borer in Colombia.</a> Cenicafe. 46, 152-163 (1995).</li><li>Bustillo, A. E., Orozco, J., Benavides, P., Portilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+and+use+of+parasitoids+for+the+control+of+the+coffee+berry+borer+in+Colombia.">Mass production and use of parasitoids for the control of the coffee berry borer in Colombia.</a> Cenicafe. 47 (4), 215-230 (1996).</li><li>Celestino, F. N., Pratissoli, D., Machado, L. C., Santos Junior, H. J. G. D., Mardgan, L., Ribeiro, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+breeding+techniques+of+the+coffee+berry+borer+[Hypothenemus+hampei+(Ferrari).">Adaptation of breeding techniques of the coffee berry borer [Hypothenemus hampei (Ferrari).</a> Coffee Science. 11 (2), 161-168 (2016).</li><li>Dom&#237;nguez, L., Parzanese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+light+in+food+preservation.">Ultraviolet light in food preservation.</a> Argentine Foods. 52 (2), 70-76 (2012).</li><li>Jaramillo, J., Chabi-Olaye, A., Poehling, H. M., Kamonjo, C., Borgemeister, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+improved+laboratory+production+technique+for+the+coffee+berry+borer+Hypothenemus+hampei,+using+fresh+coffee+berries.">Development of an improved laboratory production technique for the coffee berry borer Hypothenemus hampei, using fresh coffee berries.</a> Entomologia Experimentalis et Applicata. 130 (3), 275-281 (2009).</li><li>P&#233;rez, J., Infante, F., Vega, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+coffee+berry+borer+(Coleoptera:+Scolytidae)+have+mutualistic+fungi.">Does the coffee berry borer (Coleoptera: Scolytidae) have mutualistic fungi.</a> Annals of the Entomological Society of America. 98 (4), 483-490 (2005).</li><li>Benavides, P., Gil, P., G&#243;ngora, C., Arcila, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+pest+management.+Cenicafe.+Manual+of+the+Colombian+coffee+grower:+Research+and+technology+for+the+sustainability+of+coffee+growing.">Integrated pest management. Cenicafe. Manual of the Colombian coffee grower: Research and technology for the sustainability of coffee growing.</a> Manizales: FNC: Cenicaf&#233;. 3, 179-214 (2013).</li><li>Bustillo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+coffee+berry+borer,+Hypothenemus+hampei+(Coleoptera:+Curculionidae:+Scolytinae),+in+Colombia.">A review of the coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae), in Colombia.</a> Revista Colombiana de Entomolog&#237;a. 32 (2), 101-116 (2006).</li><li>New Chemical Control Alternative for the Integrated Management of the Coffee Berry Borer. National Coffee Research Center (Cenicaf&#233;) Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/handle/10778/557">https://biblioteca.cenicafe.org/handle/10778/557</a> (2015)</li><li>Tapias, L., Martinez, C., Benavides, P., Gongora, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+method+to+evaluate+the+effect+of+insecticides+on+the+coffee+berry+borer.">Laboratory method to evaluate the effect of insecticides on the coffee berry borer.</a> Cenicaf&#233;. 68 (2), 76-89 (2017).</li><li>Bustillo, A. E., Mar&#237;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+reactivate+the+virulence+of+Beauveria+bassiana+to+control+the+coffee+berry+borer.">How to reactivate the virulence of Beauveria bassiana to control the coffee berry borer.</a> Manejo Integrado de Plagas. 63, (2002).</li><li>Constantino, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=morphological+and+genetic+aspects+of+Hypothenemus+obscurus+and+Hypothenemus+hampei+(Coleoptera:+Curculionidae:+Scolytinae).">morphological and genetic aspects of Hypothenemus obscurus and Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae).</a> Revista Colombiana de Entomolog&#237;a. 37 (2), 173-182 (2011).</li><li>Estrela, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+action+of+sodium+hypochlorite.">Mechanism of action of sodium hypochlorite.</a> Brazilian Dental Journal. 13 (2), 113-117 (2002).</li><li>Diffey, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solar+ultraviolet+radiation+effects+on+biological+systems.">Solar ultraviolet radiation effects on biological systems.</a> Physics in Medicine and Biology. 36 (3), 299-328 (1991).</li><li>. Integrated Management of the Coffee Berry Borer: Hypothenemus hampei Ferrari in Colombia Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/hangle/10778/848">https://biblioteca.cenicafe.org/hangle/10778/848</a> (1998)</li><li>Portilla, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+artificial+diet+for+the+rearing+of+Hypothenemus+hampei.">Development and evaluation of an artificial diet for the rearing of Hypothenemus hampei.</a> Cenicaf&#233;. 50, 24-38 (1999).</li><li>Portilla, R. M., Streett, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+techniques+for+automated+mass+production+of+Hypothenemus+hampei+on+the+modified+Cenibroca+artificial+diet.">New techniques for automated mass production of Hypothenemus hampei on the modified Cenibroca artificial diet.</a> Cenicaf&#233;. 57, 37-50 (2006).</li></ol>

In this protocol, disinfection of the fruits as well as the insects are critical steps. When fruits from the field are used in the laboratory, they frequently show high contamination and dehydration since microorganisms and mites are present in the epidermis7,15,16. Therefore, using fruits or insects that are not disinfected will cause insect death due to contamination caused by microorganisms, such as bacteria or fungi, thus in...

The coffee berry borer (CBB), Hypothenemus hampei, was first detected in 1988 in Colombia and has since become the most important pest species of the coffee crop. CBB females leave the natal fruit already fertilized, seeking new fruits guided by the volatile chemicals that they emit1,2. A complete cycle is fulfilled within 23 days3 at a temperature of 25 &#176;C. The cycle starts with the founder female penetrating the seed and laying eggs in the fruit endosperm. The eclosed larvae eat the seed. If the fruits are dissected at this point, it would be possible to observe both the founder female and her offspring. After 14&#160;days, the larvae become pupae-generally, the pupae stage lasts 5 days. In the adult stage, the females copulate with their siblings, and the newly fertilized females fly away from the damaged fruits looking for new coffee fruits to start a new cycle4.
Both the penetration process and the result of larval feeding damage the coffee seed, decreasing the quality of the coffee beverage and significantly reducing the revenue; greater than 5% infestation in coffee plantations is generally considered the economic threshold.
CBB control is based on an integrated pest management (IPM) strategy, including cultural control and agronomic practices, natural biological agents, and the use of chemical insecticides, which requires safety conditions and timely application4.
To evaluate new insecticides for the control of the CBB, low-cost methodologies are needed that allow rapid results to be obtained. Both laboratory and field procedures are currently in use, including artificial diets containing coffee in which the insecticides are incorporated5,6,&#160;or spraying the insecticides on dry parchment coffee7,8,9. In addition, experiments carried out in the field using coffee tree branches covered with entomological sleeves have been reported10,11; however, these methods require intense labor and long evaluation periods.
A condition resembling natural field conditions, that is also fast and inexpensive, is the use of green or ripe coffee fruits. However, these fruits must be maintained under conditions suitable for developing the CBB, avoiding alterations and contaminants by microorganisms to maintain their quality and properties. To this end, different disinfectants have been used, as well as procedures involving heat and radiation7,9,12,13,14,15,16.
Additionally, the methods for insecticide evaluation against the CBB require simulations of adult females flying in search of fruits or penetrating those fruits17,18. For this, artificial fruit infestations have been carried out in the field8,11,19, although this process is labor-intensive and depends on environmental conditions.
Here, we describe a standardized methodology for the evaluation of products that can have different effects on the CBB under controlled environmental conditions that resemble field conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beaker with spout, low form 500 mL</td>
			<td>BRAND PP</td>
			<td>BR87826</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Shaker</td>
			<td>New Brunswick Scientific Innova 4000 Incubator Shaker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dishwashing liquid soap-AXION</td>
			<td>Colgate-Palmolive</td>
			<td>AXION</td>
			<td></td>
		</tr>
		<tr>
			<td>Hood; Horizontal Laminar Flow Station</td>
			<td>Terra Universal</td>
			<td></td>
			<td>&#160;Powder-Coated Steel, 1930 mm W x 1118 mm D x 1619 mm H, 120 V (https://www.terrauniversal.com/hood-horizontal-laminar-flow-station-9620-64a.html)</td>
		</tr>
		<tr>
			<td>Insects CBB</td>
			<td>BIOCAFE</td>
			<td></td>
			<td>(http://avispitas.blogspot.com/p/biocafe.html).</td>
		</tr>
		<tr>
			<td>Multi Fold White paper towels</td>
			<td>Familia</td>
			<td>73551</td>
			<td></td>
		</tr>
		<tr>
			<td>Preval Spray unit</td>
			<td>&#160;Preval Merck</td>
			<td>Z365556-1KT</td>
			<td>https://www.sigmaaldrich.com/CO/es/product/sigma/z365556?gclid=Cj0KCQiAweaNBhDEARIsAJ 
			5hwbfZOy1TWGj6huatFtRQt 
			AzOyHe5-oBiKnOUK2T1exuuk 
			WwJLdvxkvsaAjoYEALw_wcB</td>
		</tr>
		<tr>
			<td>Reversible Racks 96-Well</td>
			<td>heathrowscientific</td>
			<td>HEA2345A</td>
			<td>https://www.heathrowscientific.com/reversible-racks-96-well-i-hea2345a</td>
		</tr>
		<tr>
			<td>Scalpel blades N 11</td>
			<td>Merck</td>
			<td>S2771-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handles N3</td>
			<td>Merck</td>
			<td>S2896-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hypochloride</td>
			<td>The clorox company</td>
			<td>Clorox</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 508</td>
			<td>https://www.zeiss.com/microscopy/int/products/stereo-zoom-microscopes/stemi-508.html</td>
		</tr>
	</tbody>
</table>

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.

The results showed that the CBB females recognized the fruits, and depending on the characteristics of the fruit surface and the emitted odors, the CBB females started to penetrate or bore the fruits within 3 h at 21 &#176;C.
The effect of an insecticide on the CBB when applied to the coffee fruits (preinfestation procedure)&#160;after 24 h and over time is shown in Figure 4. The two insecticides (alkaloid emulsion at 5% and 6%) caused high insect mortality on Day...

Watch this Scientific Journal Video about Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei at JoVE.com

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.

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 ...

This method uses green coffee fruits to test the toxicity of insecticides against the coffee berry borer. The insecticide application is performed before or after the insect infestation to study the insect mortality repellency and other effects. Using real green coffee with nutrients adequate for insect growth is the most appropriate way to evaluate the toxicity of compounds to insects under simulated natural conditions.The protocol allows the evaluation of the toxic effects of chemical insecticides and toward pathogens such as fungi or repellent substances. This infection of the fruits and the insects is the critical step as improper disinfection can cause insect death due to contamination. Insect quality is also important as weak insects will overestimate the results of a toxic product.Visual demonstration allows identification of the appropriate stage of the fruits the insect infestation process, the appropriate moment to spread the toxic substance. And the way for dissecting the fruits. Demonstrating the procedure will be Claudia Martinez, a research assistant from the laboratory.To begin, collect the green coffee fruits early in the morning with the approximate developmental age of 120 to 150 days after flowering from coffee plantations. Select around 300 uniformly-sized and healthy fruits and withdraw the peduncles. Dip the green coffee fruits into a 2%soap solution and clean thoroughly by rubbing them.Then rinse the fruits three times with clean water. Immerse them in 0.5%sodium hypochlorite solution and place them on the shaker for 10 minutes at 110 revolutions per minute. Rinse the fruits with clean water three times and dry them with sterile paper towels.Irradiate the fruits by placing them at 55 centimeters from the UV source for 15 minutes, inside a UV-enabled horizontal laminar flow station. Move the fruits after every five minutes to ensure the proper irradiation of the whole fruit. To set up the bioassays, immerse the coffee berry borers that emerged on the same day in 0.5%sodium hypochlorite solution and agitate them slowly with a brush for 10 minutes.Filter these insects through a muslin cloth and wash them three times with sterile distilled water. Remove excess water with sterile paper towels. Prepare a group of 30 green coffee fruits per experimental unit and place them in a plastic box.Apply different concentrations of a test product for evaluation using a portable sprayer unit, here, 5%and 6%alkaloid emulsions were used. As a control, spray a group of green coffee fruits with water. Utilize at least three experimental units per treatment, spraying one after another.In a sterile hood, release two coffee berry borer adults per green coffee fruit, and cover the boxes after 30 minutes. Leave the plastic boxes with the infested green coffee fruits in the dark inside a room or incubator, having controlled conditions. After 1, 7, 15, and 21 days, count the number of borer fruits and living and dead insects outside the fruits in each box.At 20 days post-infestation, dissect each green coffee fruit under a stereo microscope with 10x magnification and count the number of healthy seeds or seeds damaged by the insects in each fruit. Count the different coffee berry borer biological stages. Observe the number of dead insects in each seed to determine insect mortality per experimental unit.In a sterile hood, release coffee berry borer adults to the previously disinfected green coffee fruits in a two-to-one ratio and allow the infestation to proceed for three hours at 21 degrees Celsius. Most green coffee fruits should be infested after three hours with the abdomen of the coffee berry borer still exposed. Select 46 infested fruits and place them in a 96-well plastic rack so that the treatment can be directly sprayed on the coffee berry borer perforating the fruit.Spray three racks at least three times for treatment, one after the other and cover the racks after 30 minutes. After 20 days, dissect the green coffee fruits under a stereo microscope at 10 x magnification and count the number of healthy seeds or seeds damaged by the insects in each fruit. Count the different coffee berry borer biological stages and the number of dead insects in each seed to determine insect mortality per experimental unit.Follow the initial steps for evaluating a product with a protective effect on the fruits and release the green coffee fruits adults into the plastic boxes. Then count the number of coffee berry borers that fly away from the boxes and the number that infest the green coffee fruits. After the incubation, under controlled conditions, followed by dissection at 20 days post-infestation and counting the seeds and dead insects, follow the steps outlined for evaluating the product, after the coffee berry borer infestation.Count the number of coffee berry bores that moved out of the green coffee fruits, or flew away from the fruits. After spraying each treatment, then follow the steps from incubation under controlled conditions to determine the insect mortality per experimental unit. 5%and 6%insecticide applied to the coffee berry borer, caused high insect mortality on day 20, and showed significant differences compared to control.It was seen that the application of insecticide two resulted in 94%of healthy seeds, and insecticide one resulted in 89%of healthy seeds. 37%of the seeds were not infested in the control group. In the case of post-infestation, the products penetrated the insect cuticle causing insect mortality.The highest mortality occurred with insecticide two. The effects of the insecticides were reflected in the percentage of healthy uninfected seeds, on day 20 of the evaluation. A reduced insect population was found inside the coffee seeds compared with the insect population found in the control group, sprayed with water.Entomopathogen and repellent combined action resulted in minimum seed damage and maximum insect mortality. This infection of the fruits and disinfection of the insects ensure the protocol success. This methodology also can be used for evaluating the tolerance or resistance of coffee fruits to the coffee berry borer.A negative effect can be also observed against the insect population in the case of insect-resistant coffee seeds. This methodology can be used not only in entomology for evaluating insecticide with different modes of action but also in plant breeding for evaluation of tolerant varieties and plant pathology to study the fruit/pathogen interaction.

methodology-to-test-control-agents-insecticides-against-coffee-berry

Research

JoVE Journal

Environment

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

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 ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

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 ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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 ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

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

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

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