In this laboratory you will inoculate maize (corn) with two different viruses and look at the interaction of these two viruses. The two viruses are maize dwarf mosaic, which is a common aphid transmitted potyvirus, and maize chlorotic mottle virus (symptoms) which is a relatively rare virus (in the U.S. it's found only in north central Kansas and south central Nebraska). Together the two viruses produce " corn lethal necrosis" which is much more severe than either virus alone. You will inoculate two different maize inbreds Va35, which is relatively resistant, and SDP2, which is especially susceptible. It will take two to three weeks to see good symptoms. You will also inoculate an unknown onto a universal host, Chenopodium quinoa.
Inoculating plant viruses
Inoculating plant viruses involves rubbing an extract of virus infected tissue on a healthy leaf. Leaves are commonly ground with a mortar and pestle. Don't take too much tissue or you'll spend excessive time grinding. Leaves can be ground in water or more commonly in dilute (< 0.05M) phosphate buffer. One mixes an abrasive (600 mesh carborundum) with the inoculum and rubs it onto the surface of a healthy leaf. Most cells on the leaf surface remain undamaged and will not be infected. On the other hand many cells will be killed. Only those cells which are damaged and recover will be infected. You must rub the leaf hard enough to damage the leaf surface, but not so hard that you seriously damage the surface.
Pot labels (one per pot)
Sharpee or crayon to mark pot labels
Phosphate buffer, pH ~7
Mortar and pestle
600 mesh carborundum
3 pots of Va35 seedlings
3 pots of SDP2 seedlings
1 pot of Chenopodium quinoa plants
MDMV infected tissue (piece of a leaf)
MCMV infected tissue (piece of a leaf)
Unknown - a leaf from the field with virus like symptoms
Begin by labeling your pots (MDMV, MCMV, Mix, include date). Grind a portion of an MDMV infected leaf in a small amount of phosphate buffer (with mortar and pestle). Add a small amount of carborundum, mix and rub leaf surfaces (inoculate both MDMV and mix). Carefully rinse your mortar and pestle and then inoculate MCMV in the same way. MCMV is much more infectious than MDMV so it is good practice to inoculate the latter first (to minimize contamination). Normally it would be good practice to have a 4th set of seedlings which one would inoculate with buffer alone.
Finding a field sample
Pick the leaf from a plant that has virus-like symptoms. Mosaic is the safest symptom since relatively few things other than viruses produce it. Clover, especially white clover, with mosaic symptoms can usually be found. Physalis subglabrata, a common weed, is often virus infected. Ajuga reptans, a common ground cover, often has virus symptoms. It is especially interesting to find potential virus symptoms on a different plant that nobody else has looked at, but this is more difficult.
Inoculating field sample
Neither MCMV nor MDMV infect C. quinoa. Therefore it is safe to use the same mortar and pestle that you used for the maize inoculations. Remove all but two quinoa plants from the pot. Mark a pot label. Trim the lower leaves from the remaining plants leaving only about 3 expanded leaves at the top. Grind your unknown and inoculate the quinoa leaves (as you did with maize). After about 30 sec rinse the leaves with cold water to wash off salts (phosphate) which could damage the leaves, particularly on sunny days. If you would like to inoculate only one plant and find another sample to inoculate in the next few days feel free. I will ultimately collect samples of viruses which show up and run them through the standard minipurification protocol..
What to observe and record
Symptoms on the maize plants will appear after about a week. Observe them every few days and record. The symptoms of your unknown on C. quinoa are less easy to predict. Observe and record every few days. Note that some viruses will give local (inoculated leaf) symptoms on quinoa and other will go systemic. Note what other people's quinoas look like. Hopefully we will find a range of viruses and see a range of symptoms.