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

The overall goal of this procedure is to quickly and reliably collect spider mites at a given developmental stage. This is accomplished by first wearing mites on bean plants. Next, the experimental plant is infested with female mites using a paintbrush or pump, depending on the number of mites to be applied to a single plant.

The final step is manual plant damage quantification using the grid method in a graphics editing program. Ultimately, statistical analysis is used to show differential susceptibility of plant lines or ecotypes to spider mite orry. The made advantage of this technique over existing methods is that it provides an efficient method of quick and easy collection of a large number of adult female mites, their application to an experimental plant host, and the assessment of plant damage due to mite feeding.

The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage that can be used for subsequent experimental application. To begin grow California red kidney bean plants from seeds for two to three weeks prior to infestation intermix these plants with infested plants. Adult mites will rapidly colonize the fresh plant material.

Remove old infested bean plants every seven to 10 days and replace them with fresh plants to collect mites using the washing method. Place a fresh bean plant in contact with infested plants one to two days prior to the experiment. Use 20 to 30 fresh bean plants to collect 1000 to 2000 adult female mites.

On the day of the experiment, cut the mite. Invested bean plants at the base, then wash the infested bean plants two to three plants at a time in a 0.001%tween 20 solution. Each plant is washed two to three times to collect all mites from the leaves.

Work within a 10 minute window to avoid mite mortality. Next, prepare a 500 micrometer sieve to remove debris and a 300 micrometer sieve to collect female adult mites. Filter the suspension through the 500 micrometer sieve, then refill the suspension through the 300 micrometer sieve.

Adult female mites are retained if too much foam has accumulated in the sieve. With mites, it can be gently washed under slow running room temperature. Tap water dip a 300 micrometer sieve with adult female mites in clean tap water to remove the tween 20 spread mites in a single layer at the bottom of the sieve.

Use a paper towel to remove excess water from the mesh and sides of the sieve, so if the mites can dry quickly in order to recover. Next place a sieve with mites on top of the sieve that is used as a support. Surround the bottom sieve with water to prevent the mites from dispersing.

This will allow the mites to move freely, but prevent them from escaping. Spider mite infestation with a brush is used for application of up to 30 mites per plant. Use a soft hair round art brush of size, zero, zero or finer.

Wet the brush with room temperature. Tap water gently pick up mites from the sieve using the brush tip and transfer them to the plant. When more than 30 mites are applied, plant infestation is achieved with mites collected by a pump vacuum line.

For this method, attach a one milliliter pipette tip to the tube that connects to the pump or vacuum line by using a 1.5 milliliter centrifuge tube. Cut at the bottom as an adapter between the pump vacuum tube and the tip place a piece of paper towel between the pipette tip and the adapter. The purpose of this is the trap mites inside the pipette tip during the aspiration process, and also to reduce the airflow, collect mites directly from the sieve using the pipette tip.

Alternatively, collect mites one by one from infested leaves using a stereoscope. When the required number of mites has been collected. Remove the pipette tip from the tube, making sure that the piece of paper towel at the back of the pipette tip is undisturbed.

Collect mites by tapping the pipette tip and clumping the mites together. Then place them on the leaf. Within seconds, the mites will start to disperse on the leaf.

To record plant damage, cover a scanner bed with a transparency sheet to prevent contaminating the scanner's surface. With plant material. Cut an entire rosette and place it on the scanner bed.

So the a axial side of the leaves are facing the scanner. Light source and capture element, multiple rosettes can be scanned simultaneously. Alternatively, if rosettes are too dense to capture individual leaves without overlap, dissect the roset into individual leaves or groups of non-overlapping leaves with fine scissors before placing them on the scanner.

Bed Cover rosettes or leaves with a piece of white paper to prevent adherence to and contamination of the scanner cover. Close the scanner cover and perform a scan with a resolution of 1, 200 DPI brightness set to plus 25 and file type set to maximum quality jpeg. Save the image files for subsequent damage analysis.

To quantify plant damage, open the image of the plant with Photoshop. Create a new layer. Then overlay the scanned image with the grid of point 25 millimeter by point.

25 millimeter divisions on an overlaid layer. Mark damaged leaf areas using a dot with the pencil tool. Use one single dot for each square of the grid below where there is damage covering more than one half of the grid unit.

The size of the. is defined in pixels. When all squares of the grid that show plant damage covering at least half of the grid unit are marked with a dot, used, a histogram tool to determine the number of pixels on the layer representing the number of dots, and by extension the total number of marked squares.

Calculate the number of dots marked by dividing the total number of pixels by the number of pixel per dot. Calculate the total area of damage by multiplying the number of marked dots on the image by the area of a square of the grid as one dot corresponds to one square of the grid. This plot displays the damage area measured on three week old AANA plants.

Four days after application of 20 female spider mites. The results of plant damage are reproducible, demonstrating that collected mites are of similar physiological state and suitable for plant spider mite interaction studies. This plot displays the damage area measured on three week old AANA plants four days after application of 20 female spider mites.

As an example of experimental application, natural variation and susceptibility to spider mite, orry was assessed across three aana ess by application of 20 female spider mites per plant using a wet brush and recording damage four days post infestation. Quantification of damage data can be presented as a bar graph or a box plot, and analyzed by the appropriate statistical method. In this example, susceptibility to spider mite or bry significantly varies across the sessions.

Isolation and infestation steps can be completed within 30 minutes depending on the number of samples and workflow choice. It's important to remember that mite should not be submerged in between 20 solution for more than 10 minutes. The isolation using seed system may be used to obtain fractions of mites at various developmental stages, which can be utilized for a variety of experiments, not only in the field of pest plant interaction, but also development physiology and molecular biology.