We appreciate the assistance of Chris Parker who daily scanned the plants and Curtis Hubbard who maintained the plants in the greenhouse.

1. Establish TSSM Colonies on Pinto Beans
<ol>
	<li>Plant pinto beans, Phaseolus vulgaris L., in plastic trays (56 x 28 x 5 cm3) containing potting soil in the greenhouse as shown in Figure 1.</li>
	<li>Label the trays with marking sticks according to treatment and replication.</li>
	<li>Set and maintain greenhouse temperature to 90 &#176;F and 70% Relative Humidity.</li>
	<li>Grow beans to 1-2 trifoliate leaf stage10 as shown in Figure 2.</li>
	<li>Collect spider mites from cotton plants infested naturally with mites by removing infested leaves.</li>
	<li>Place spider mite infested cotton leaves on pinto beans as often as necessary until all plants in the trays are infested with numerous TSSMs.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54314/54314fig1.jpg" /> 
Figure 1: Planting pinto beans in plastic trays. Pinto beans seeds were planted in plastic trays (56 x 28 x 5 cm3) in the greenhouse and were watered daily. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54314/54314fig2.jpg" /> 
Figure 2: Pinto beans with trifoliate leaves. The first true leaf that is formed after the cotyledons emerge from the soil is the simple or unifoliate leaf. Subsequent leaves are the trifoliate leaves with denticulate tips. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Transfer TSSM to Cotton Plants
<ol>
	<li>Grow Non-Bt (not genetically modified) cotton plants to 4-5 true leaf stage in plastic trays (56 x 28 x 5 cm) in the greenhouse as specified in step 1.3 and shown in Figure 3.</li>
	<li>Transfer spider mite colonies from pinto beans onto young cotton plants at 4-5 true leaf stage.
	<ol>
		<li>Transfer 3 masses of spider mites for lightly infested plants. Note: At very high infestation levels, spider mites form masses or bolls11 and are found hanging on leaf tips as seen in Figure 4.
		<ol>
			<li>Place a pan under pinto bean leaf tip containing TSSM masses.</li>
			<li>Cut pinto bean leaf tips with scissors, allowing TSSM masses to fall into the pan as shown in Figures 5 and 6.</li>
			<li>Turn pan upside down over cotton plants and tap TSSM masses onto cotton plants grown in plastic trays as shown in Figure 7. Note: Each tray contained ~100 cotton plants.</li>
			<li>Randomly spread 3 masses of TSSM onto cotton plants.</li>
		</ol>
		</li>
		<li>Transfer 20 masses for medially infested plants.
		<ol>
			<li>Place a pan under pinto bean leaf tip containing TSSM masses.</li>
			<li>Cut pinto bean leaf tips with scissors, allowing them to fall into the pan.</li>
			<li>Collect 20 masses in the pan.</li>
			<li>Turn pan upside down over cotton plants and tap out TSSM masses onto ~100 cotton plants raised in the greenhouse.</li>
		</ol>
		</li>
		<li>Transfer 40 masses for heavily infested plants.
		<ol>
			<li>Place a pan under pinto bean leaf tip containing TSSM masses.</li>
			<li>Cut pinto bean leaf tips with scissors, allowing them to fall into the pan.</li>
			<li>Collect 40 masses in the pan.</li>
			<li>Turn pan upside down over cotton plants, tap out TSSM masses onto ~100 cotton plants and spread them randomly.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/54314/54314fig3.jpg" /> 
Figure 3. Cotton plants with 4-5 true leaf stage. The cotyledons emerge from the soil as leaf-like structures oriented opposite to each other on the seedling stem. The apical meristem emerges through the cotyledons and form the first true leaves. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54314/54314fig4.jpg" /> 
Figure 4. TSSM masses hanging on a trifoliate bean leaf. TSSM lives in colonies and when populations reach high density, they form masses or boll-like structures and congregate at the leaf tips for dispersal. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54314/54314fig5.jpg" /> 
Figure 5. Cutting pinto bean leaf tips containing a TSSM mass with scissors. Trifoliate bean leaf tips containing TSSM masses were removed with scissors for infesting cotton plants. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54314/54314fig6.jpg" /> 
Figure 6. TSSM masses on bean leaf tips placed inside the pan. When sufficient number of trifoliate bean leaf tips with TSSM were found in the test plants, they were removed and placed inside the pan. These samples were used to infest treatment categories: light, medium and heavy which received 3, 20 and 40 masses of TSSM, respectively. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54314/54314fig7.jpg" /> 
Figure 7. Turning pan upside down. The pans containing trifoliate bean leaf tips with TSSM were turned upside down on cotton canopy to infest the test plants. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Scan TSSM Infested Cotton Plants with the Multispectral Optical Sensor
<ol>
	<li>Horizontally mount the optical sensor onto the greenhouse frame approximately 7 ft above the floor as shown in Figure 8. Set the distance between the scanner and the plant canopy at 36&#34;. Use a carpenter&#39;s level to ensure that the sensor is horizontally level.</li>
	<li>Place uninfested trays of cotton plants on a wheeled push cart.</li>
	<li>Activate the sensor switch and slowly push the cart under the sensor until the tray completely passes the sensor head as shown in Figure 8. Turn off switch. Retract the cart.</li>
	<li>Repeat Step 3.3 three times, for a total of 3 replications.</li>
	<li>Repeat this procedure for all trays of cotton.</li>
	<li>Repeat scanning on Day 1, Day 5, Day 6, Day 7, Day 9, Day 10, Day 12, Day 13 and Day 14 after treatment (DAT). The scanning provided the NDVI (Normalized Difference Vegetation Index) values12. Transmit the NDVI values to and store on a pocket PC which then can be downloaded to a computer in text format. 
	NOTE: NDVI was calculated from the following equation: NDVI = (NIR - RED)/(NIR + RED), where RED and NIR are the spectral reflectance values (0-255) in the red and near-infrared spectrums at 660 and 770 nm, respectively.</li>
</ol>
<img alt="Figure 8" src="/files/ftp_upload/54314/54314fig8.jpg" /> 
Figure 8. A multispectral optical sensor used to quantitatively measure the health of cotton plants infested with varying density levels of TSSM. The wheeled push cart with the test plants were slowly traversed under the sensor head to obtain spectral reflectance values. a is the sensor head; b is pocket PC; c is battery compartment and input/output ports; d is RS-232 serial data cable and e is the craft paper to provide uniform background. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Data Analyses
<ol>
	<li>Obtain the maximum NDVI values using the Proc Means procedure13. Calculate the percent reduction in NDVI values for each day of observation using Day 0 as a reference value. Analyze the data using the repeated measures PROC GLM procedure13. 
	NOTE: Means were separated using the Duncan&#39;s Multiple Range Test at P = 0.05. Means with the same lower case letters were not significantly different.</li>
	<li>Perform graphical illustrations of the data14 as shown in Figure 9.</li>
</ol>
<img alt="Figure 9" src="/files/ftp_upload/54314/54314fig9.jpg" /> 
Figure 9. Percent reduction or change in NDVI relative to days after treatment. JMP software was used to graphically illustrate the functional relationship between percent change in NDVI relative to days of sampling (DAT). <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54314/54314fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

Conventionally, insecticide efficacy tests conducted in the field include several treatments of the chemical applied at different rates and compared to an untreated check. Acaricides with varying toxicity profiles against instar and adult stages of TSSM are assessed to determine whether the damage caused by them could be reduced by the chemical treatment. TSSM samples are collected and brought to the laboratory where they are examined under the microscope and the varying stages of TSSM are counted and recorded. It is critically important to take adequate samples of the host plant to determine the damage in each treatment and differentiate them with statistically acceptable precision. The number of samples that are required to differentiate the treatments from each other depends upon the distribution profile of the organism. Highly patchy distribution of the TSSM leads to a considerable amount of variation between sample areas, and many plants must be sampled in order to ensure reproducibility of the population estimates. However, budget, manpower, time and statistical precision are important factors that impinge upon sampling techniques. It behooves the researcher to allocate the available resources optimally to conduct sampling with the least cost but with most precision.
Entomologists in lieu of counting TSSM stages visually score the damage based upon a scale of no damage to varying levels of damage. For instance, some researchers proposed binomial sampling, where only the proportion of infested leaves was scored rather than the number of spider mites per leaf9,19. Others estimated damage by TSSM on cotton based on a leaf reddening index scale, which varied from stippling and reddening to extensive reddening of vegetation canopy19. These methods are arbitrary, anecdotal and biased by individual perceptions of the degree of damage. A more robust and quantitative assessment of the damage caused by TSSM is required to evaluate and separate the treatments with statistical precision.
The ground-based multispectral optical sensor appears to be an improved sampling tool to quantitatively determine the damage inflicted by TSSM and to separate the treatments more precisely than the visual damage scoring system adopted by many researchers. However, researchers have reported that the data intensive hyperspectral remote sensing provided numerous spectral signatures to identify and detect crop stresses and canopy characteristics compared to multispectral remote sensing which is less data intensive with two wave lengths20,21. Using a hyperspectral spectrometer, Reisig and Godfrey found that the NIR reflectance wavelength &#8776;850 nm as an informative spectrum in distinguishing arthropod-infested from uninfested cotton22. In this study, we have shown that the multispectral reflectance values (NIR value being &#8776;770 nm) with just two spectral bands were able to identify and characterize cotton plants infested with varying densities of TSSM. Also, we have reported earlier that the multispectral optical sensor not only effectively separated cotton plants infested with widely varying density categories of TSSM, but also showed that spiromesifen was more effective than abamectin in controlling TSSM in early season cotton in the greenhouse at one-half rate of the lowest label rate23.
The multispectral optical sensor could be mounted on a mobile research platform and the reflectance values could be obtained from the treated areas of the vegetation canopies without human subjectivity. Acaricidal efficacy data can thus be obtained without much human labor. The NDVI data can be easily uploaded to the computer and analyzed using commercial statistical software. A GPS receiver also can be mounted onto the mobile platform to collect the GPS coordinates in order to generate a damage map of the field. Using multispectral reflectance signatures from the plant canopy, the multispectral optical sensor provides a rapid and cost effective means of identifying and quantifying plant stress. Moreover, a much larger area of the field can be covered in less time with higher spatial resolution of plant canopy compared to conventional field scouting. It is important to remember that the threshold damage level for TSSM on cotton is variable from region to region in the United States. For example, TSSM damage would be higher in an arid environment such as in California compared to the Midsouth region where rainfall and high humidity often prevail24. Therefore, yield loss due to TSSM damage will be variable and so is the damage threshold. However, reports from Mississippi, Arkansas and Tennessee reveal that the threshold level for TSSM on cotton appears to be when 30 to 50% of the plants are infested and populations are actively increasing5. Furthermore, field grown plants are exposed to multiple stresses including water stresses and feeding of arthropod herbivores and the interactions between these activities can substantially reduce plant productivity and are likely to influence damage threshold. The TSSM can reduce stomatal conductance, photosynthesis and transpiration rate in cotton25. Plants grown in the greenhouse are influenced by UV light radiation and it does significantly influence stomatal function, photosynthesis and canopy morphology26,27 and could probably have an additive effect on plant stress. However, TSSM is capable of avoiding UV radiation by having access to habitat protected from solar radiation on the lower surface of the plant canopy28,29,30, where it resides.
The height of the optical sensor above the target canopy and the orientation of the sensor relative to the target are important factors that affected significantly the reflectance values obtained by the multispectral optical sensor31. For instance, when the mobile research platform traverses through a row crop field such as when cotton canopy is open, the sensor is likely to produce different results depending upon the orientation of the sensor, either parallel to or perpendicular to the row. It is also likely that soil and other background materials may greatly influence the sensor readings, especially when the sensor is oriented perpendicular to the row. In order to obtain maximum response from the sensor, the sensor head should be oriented in-line with and directly above the rows. Although orienting the light beam perpendicular to the rows is more likely to pick up background soil reflectance, this may be acceptable, however, when cotton canopy is closed with lush vegetation. In addition, operators should follow manufacturer&#39;s recommendations on an operating height range of 81-122 cm and orienting the sensor head in-line with the target to obtain maximum signal response. It is important to charge the sensor battery before use or it should be kept plugged in to avoid troubleshooting. Low battery level is likely to produce erroneous readings.

Two-spotted spider mite, Tetranychus urticae (Koch) is a polyphagous and cosmopolitan pest of many field and horticultural plants1,2. It lives inside webbings in colonies on the bottom surface of the plant3,4. It has evolved from being a late season to an early season pest in the mid-southern United States over the last decade5. TSSM was the 5th most damaging pest of cotton and caused an estimated loss of 57,441 bales of cotton and 0.167% reduction in yield in the United States in 20116,7. Its short life cycle, high fecundity and haploid-diploid sex determination combined with the ability to digest and detoxify xenobiotics have exacerbated the development of resistance to pesticides8. Presently, acaricides remain as the only dependable control mechanism for the suppression of T. urticae. Therefore, field entomologists constantly evaluate currently available and newly developed acaricides for efficacy.
The estimation of damage by the spider mites is usually conducted by scoring the damage on a subjective scale because of the difficulty encountered in manually counting the mites. Some conducted binomial sampling, where only the proportion of infested leaves was scored rather than the number of spider mites per leaf9. A leaf reddening index scale, which varied from stippling and reddening to extensive reddening of the vegetation, was used as a criterion in estimating damage. The spatial distribution pattern of T. urticae on cotton conformed to a clumped distribution pattern9. Mites are distributed on cotton foliage from sparse to heavily clustered and remain so under field condition. Such distribution pattern coupled with its small size, mobility and prolific reproduction makes enumeration of TSSM difficult. Reliable alternative techniques are needed for the assessment of mite density in order to quantitatively evaluate efficacy of acaricides against TSSM.
The objective of this study was to separate cotton plants damaged by varying densities of TSSM by using an optical multispectral sensor. Our intent was to determine if the ground-based optical sensor could classify and separate healthy cotton plants from those damaged by the spider mites.

<table><tbody><tr><td>GreenSeeker</td><td>Trimble Ag. Division&lt;br /&gt;Westminster, CO</td><td>Model 505</td><td>Red NDVI sensor</td></tr><tr><td>Pinto beans</td><td>Producer&#039;s Co-op., Bryan, TX</td><td>Not applicable</td><td>Free choice item</td></tr><tr><td>Deltapine cotton seeds</td><td>Brazos Bottom Crop Care, Caldwell, TX77836</td><td>Not applicable</td><td>436 RR; NonBt &amp;amp; RoundUp Ready</td></tr><tr><td>Plastic trays</td><td>BWI, Schulenberg, TX</td><td>FG1020NL7</td><td>56 cm x 28 cm</td></tr><tr><td>Label sticks</td><td>Gempler&#039;s, Janesville, WI 53547</td><td>Item # 151276</td><td>Durable spike-style pot markers</td></tr><tr><td>4-wheel Garden push cart</td><td>Farm Tek, Dyersville, IA 52040</td><td>Item # 108676</td><td>61 cm x 122 cm</td></tr></tbody></table>

remote sensing evaluation of two-spotted spider mite damage on greenhouse cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

The sensor emits red and infrared light and this light in turn gets reflected back from the plant canopy. The reflected light serves as a quantitative measurement of the visible and the near-infrared bands of the electromagnetic spectrum and is numerically recorded as NDVI (Normalized Difference Vegetation Index) reading. The NDVI values vary from 0 to 0.99. The higher the NDVI reading the healthier the plant canopy. Healthy vegetation absorbs visible light and reflects the near-infrared light and unhealthy vegetation reflects more visible light and less near-infrared light. NDVI serves as a surrogate to photosynthetic activity and this spectral property is highly correlated to photosynthetically active radiation15,16,17,18. Very low values of NDVI (0.1 and below) corresponds to barren areas of rock or sand, while moderate values (0.2 to 0.3) represent shrub and grassland and high values (0.6 to 0.8) indicate temperate and tropical vegetation. The reflectance measurements were obtained under daytime lighting in the red and near-infrared regions of the spectra.
NDVI was calculated from the following equation: NDVI = (NIR - RED)/(NIR + RED), where RED and NIR are the spectral reflectance values (0-255) in the red and near-infrared spectrums at 660 and 770 nm, respectively. The sensor records reflectance values every 100 msec. The maximum NDVI readings culled from an array of NDVI values taken each time by the sensor were used in the analyses to minimize background reflectance and to provide consistently repeatable numerical values.
The analysis of variance of the data revealed that significant differences in percent reduction in NDVI were observed between lightly, medially and heavily infested cotton plants compared to the untreated control (F = 436.4; P &#60;0.0001; df = 3, 32). The NDVI values which described plant vigor during the test period varied significantly between days of observation (F = 1398.2; P &#60;0.0001; df = 8, 256). Also, the percent reduction in NDVI values were inversely correlated to the treatments during the observation period (DAT), but seldom tended to deviate from this pattern and interacting significantly with DAT (F = 201.5; P &#60;0.0001; df = 24, 256). The MANOVA test criteria for no DAT effect was significant as well (Wilk&#39;s &#955; = 0.00913; F = 339.0; P &#60;0.0001; df = 8, 25). Similarly, the interaction between DAT and treatment was significant (Wilk&#39;s &#955; = 0.00101; F = 29.8; P &#60;0.0001; df = 24, 73).
Figure 9 shows percent change in plant vigor as demonstrated by NDVI values during the observation period. A positive percent change in NDVI value indicates healthy growing plants, while a negative value indicates that the vigor of the plant has decreased since the first measurement (i.e. Day 0) was made. The non-infested control plants showed increased vegetative growth throughout the course of the study, while the TSSM-infested plants showed degradation in health over time. Mean separation of the treatments shown in Table 1 reveals that no definable difference in percent reduction in NDVI between treatment categories (light, medium and heavy) and the control was observed until Day 5 when infestation classes significantly deviated from the control and remained overwhelmingly so thereafter. This data demonstrates that the optical sensor can be effectively used in lieu of labor-intensive manual sampling to assess treatment efficacy against acaricides on cotton.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="10">Days After Treatment (DAT)</td>
		</tr>
		<tr>
			<td>Infestation Category</td>
			<td>1</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>9</td>
			<td>10</td>
			<td>12</td>
			<td>13</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>1.18 &#177;0.33a</td>
			<td>2.70 &#177;0.40a</td>
			<td>4.0 &#177;0.36a</td>
			<td>3.94 &#177;0.37a</td>
			<td>3.68 &#177;0.53a</td>
			<td>2.57 &#177;0.42a</td>
			<td>2.96 &#177;0.47a</td>
			<td>3.48 &#177;0.38a</td>
			<td>3.08 &#177;0.22a</td>
		</tr>
		<tr>
			<td>Light</td>
			<td>-0.13 &#177;0.13b</td>
			<td>-0.71 &#177;0.29b</td>
			<td>-0.65 &#177;0.28b</td>
			<td>-2.02 &#177;0.47b</td>
			<td>-5.68 &#177;0.72b</td>
			<td>-11.17 &#177;0.94b</td>
			<td>-15.73 &#177;1.76b</td>
			<td>-19.54 &#177;1.68b</td>
			<td>-24.9 &#177;1.90b</td>
		</tr>
		<tr>
			<td>Medium</td>
			<td>-1.83 &#177;0.42c</td>
			<td>-7.06 &#177;0.63c</td>
			<td>-9.61 &#177;0.53c</td>
			<td>-10.39 &#177;0.57c</td>
			<td>-17.06 &#177;0.80c</td>
			<td>-26.92 &#177;0.72c</td>
			<td>-33.84 &#177;0.96c</td>
			<td>-37.05 &#177;1.14c</td>
			<td>-41.74 &#177;0.73c</td>
		</tr>
		<tr>
			<td>Heavy</td>
			<td>-0.97 &#177;0.58bc</td>
			<td>-11.76 &#177;0.29d</td>
			<td>-13.83 &#177;0.86d</td>
			<td>-15.20 &#177;0.63d</td>
			<td>-25.0 &#177;1.0d</td>
			<td>-34.63 &#177;0.54d</td>
			<td>-39.07 &#177;0.94d</td>
			<td>-42.68 &#177;0.62d</td>
			<td>-46.71 &#177;0.63</td>
		</tr>
	</tbody>
</table>
Table 1: Percent reduction in Max NDVI after cotton plants were infested with varying number of clusters or masses of TSSM. Cotton plants grown in plastic trays in the greenhouse were infested with three categories of spider mite density. Category Light received 3 masses or clusters of TSSM per tray, Category Medium received 20 masses per tray and Category Heavy received 40 masses per tray. Means were separated from control according to Duncan&#39;s Multiple Range Test (P = 0.05). Means followed by the same lower case letter were not significantly different at the 5% level of probability.

Watch this Scientific Journal Video about Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton at JoVE.com

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

remote-sensing-evaluation-two-spotted-spider-mite-damage-on

Nanyang Technological University

Research

JoVE Journal

Environment

8.5K Views.  USDA-ARS, SPARC-AATRU. This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

Video: Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

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 ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

Genetics

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of different gall types and prevents the development of normal shoots. Repeated and uncontrolled attacks cause, besides the production of galls and the attendant gall-related reduction in leaf area, progressive corruption of the branch architecture, including the death of branch parts, and an increase in dormant bud activation. Thus far, there have been few attempts to quantify branch architecture damage. Further, the different methods for assessing infestation degree (MAID) that have been developed focus only on the galls' presence and abundance.
Using the leaf area to sapwood area relationship as a green biomass indicator, we developed in a previous study a damage composite index (DCI) that takes into account the most important branch architectural features, allowing for realistic damage assessment during the entire epidemic process. 
The aim of this study is to present this novel method and highlight differences in the damage description with respect to other broadly used indices. Results show how the DCI depicts branch damage better, especially during the epidemic peak, compared to MAID, which tend to underestimate it. We conclude by suggesting how to properly evaluate the overall impact of the pest by means of our composite damage index, the infestation degree using classic methods, and crown transparency evaluations.

Evaluating Dryocosmus Kuriphilus-induced Damage on Castanea Sativa

It is common practice to assess the damage caused by Dryocosmus kuriphilus by considering the abundance of galls alone rather than by also taking related branch corruption into consideration. We propose a composite damage index that takes into account the most important branch features, thus enabling more realistic damage assessment.

Dryocosmus kuriphilus Yasumatsu has become a major pest for Castaneasativa since its arrival in Europe. Its galling activity results in the formation of ...

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 ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Biochemistry

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Immunology and Infection

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton