In this photograph of carrots, the root galls formed as a result of infestation by root knot nematode can easily be seen. Each gall contains one or more adult female nematodes.
Here you see the adult female feeding on a root. Her head is within the vascular tissue of the root and a large egg mass can be seen at the posterior end of the body which is to your left. Identification of root knot nematodes to species has traditionally been accomplished by cutting off the posterior end of the adult female....
and mounting the cuticle on a glass slide for observation under a compound microscope at approximately 1,000 times magnification, as you see here. The perineal patterns of four different species of root knot nematode are shown in this picture. To confidently and accurately identify these patterns takes considerable practice over a long period of time.
In automated isozyme electrophoresis, a PhastSystem machine is used to pass electric current through disrupted individual female nematodes, separating their proteins into bands. Specific proteins can be visualized with chemical stains......
as you see depicted in this chart. From left to right are the isozyme patterns of the most common species found in California: arenaria, incognita, hapla, javanica, chitwoodi and naasi. To make them visible, the bands of two different enzymes, MDH or malate dehydrogenase; and esterase have been stained.
This is the separation compartment of the PhastSystem where 2 polyacrylimide gels (called PhastGels), each containing up to 12 nematodes, can be electrophoresed at the same time. Using forceps, we place a gel onto the cooling surface of the compartment.
Next, we place the buffer strip holder over the gel, put buffer strips at each end of the gel and lower the electrode assembly and sample applicator arms.
The next step is similar to perineal pattern preparation. Individual adult females are dissected from plant roots.
They are placed individually into depressions on a small square of black plastic called the sample-well stamp. Each nematode is then macerated with the tip of a plastic toothpick.
A sample applicator which has twelve individual protruberances is pressed into the wells.
The material which is picked up on the tips of the applicator is transferred to a gel.
This is a close up of the control panel of the PhastSystem apparatus. We will use only a few of the buttons available. Most of them are used to program the machine when it is first received. We press several buttons in series and then electric current passes through the gel from the lower to the upper end for one hour and as it does so, it separates the protein from the nematodes into bands.
While this process is occurring, we prepare the two stains which will make the protein bands visible.
After an hour. we remove the gel from the machine and stain it in a small plastic box.
The protein bands are then visible and we can compare them with the chart of known patterns to determine the species present.
The PCR DNA technique analyzes the genetic material of the nematode. It requires less starting material, and so can be used to identify the species of individual larvae or eggs. For this demonstration, we obtain root knot larvae from a hydroponic tomato culture maintained to produce large quantities of inoculum for experiments. The root system of one hydroponic plant can produce up to a million larvae a day.
A stainless steel needle is used to pick up a single juvenile, place it in the bottom of a micro-centrifuge tube, and then macerate it against the sides of the tube to release the genetic material.
Next a small amount of water is added to be tube, followed by microliter quantities of a mixture of commercially available materials needed to multiply DNA. These include an enzyme to catalyze the reaction, nucleotides which are the building blocks of DNA, a primer to serve as a template for the reaction and a buffer to keep the reaction at an optimal pH.
Next the materials are mixed, then centrifuged to be sure they are in the bottom of the tube, and a layer of oil is applied to keep the materials from refluxing in the tube during the reaction.
The next step is to turn on the Polymerase Chain Reaction orPCR machine which provides the conditions necessary to multiply the nematode DNA.
The tubes are placed into the heating and cooling block of the machine which can multiply the DNA from up to 48 individual nematode larvae simultaneously.
We then press a series of buttons to activate a preprogrammed heating and cooling cycle. For root knot nematode, the machine runs through the following cycle 35 times: First heating to 94 C for 1 minute to denature the nematode DNA, then down to 50 C so the primer can aneal to the DNA template, then up to 68 C which is the optimum temperature for the enzyme to perform the multiplication reaction. After pressing the start button on our PCR machine, it takes approximately three hours to complete this cycle of DNA multiplication. Newer machines can complete the cycle in about one hour.
After the reaction is completed, electrophoresis is conducted similar to that done in the first technique except that an agarose gel is used rather than polyacrylamide. A liquid buffer is used rather than the buffer strips.
The contents of each vial to which a blue stain has been added so its progress through the gel can be monitored, is pipetted into a single well in the gel. The electric current is turned on, then over time, the current separates the DNA molecules according to size.
The gel is then removed from the machine, stained to make the DNA bands visible, and then photographed. There is also potential for using this technique to distinguish between species of lesion and other important California nematodes.