Lauren Kalns's PLPA 631 Lab Notes: September 26 Lab

The goals for this lab were to:

Observe ascospores, microconidia, and macroconidia on Neurospora
Cross Neurospora strains
Work with the Basidiomycete Ustilago maydis to infect health corn plants, learn the life cycle, and begin to observe overtime the symptoms that appear on infected corn plants
Look at dimorphism in the Zygomycete, Mucor rouxii

Ascospores, microconidia and macroconidia of Neurospora

Three types of media were used to produce each of the spore types. A nutrient-poor medium in a petri dish produced microconidia, nutrient-rich medium in a petri dish produced macroconidia, and media contained in a microcentrifuge tube produced the ascospores. To collect macroconidia, I gently scraped a small portion of the growing fungus from the agar and placed this on a slide with a water drop. A cover slip was placed on top of this. You can see the macroconidia in the following picture.

Neurospora macroconidia

To collect the ascospores, I used a sterile loop that had been dipped in sterile water. I then took this look and gently collected material that I thought would have spores. This was placed on a slide with a drop of water in the center, and then a coverslip was placed on top of this.

Neurospora ascospores.

I was not able to get a good picture of the Neurospora microconidia, and my drawing does not provide a lot of detail. Hopefully someone else in the lab will have a picture on their blog.

Crossing of Neurospora strains

There were several strains of Neurospora available in the lab to cross. I chose SMRP11 and SMRP10 which are two opposing mating types. To do this cross, I simply took (under sterile conditions) an agar plug from each of the stock plates and then placed them on opposite ends of a clean petri dish with SC solid media. I sealed the dish in parafilm and then place this in a clear plastic box in the back of the lab. I will look at this next week to see if I have had a successful cross.

What my Neurospora cross looks like immediately after the agar plugs were placed in the plate.

Dimorphism in the Zygomycete, Mucor rouxii

In this lab we attempted to observe dimorphism of Mucor rouxii, which under aerobic conditions takes on a hyphal form and when under anaerobic conditions takes on a yeast form. Small cups with media had been previously inoculated with this fungus for our use. A small sliver of agar was cut from the center of the cup, making sure to include agar that both had access to oxygen on the surface, but that also lacked oxygen from the bottom of the cup. Ideally you should be able to see a gradient from top to bottom of the two forms, however I found that I needed to pick and choose through the top to get hyphal images, as well as from the bottom for yeast forms. Eventually I was able to successfully get a few images to demonstrate this dimorphism.

Mucor rouxii hyphal form taken from near the surface of the agar.

If you look closely near the cut-off end of this image you can see the formation of the yeast type cells of Mucor rouxii.

Also in the Mucor cups I was able to see many of these round, brown sporangiophores.

Infection of corn plants with Ustilago maydis

To learn more about the infection process and life cycle of a Basidiomycete, we began today the infection process of corn by Ustilago maydis. This fungus causes corn smut, which will create galls on the leaves and reduce yields. To infect our clean, healthy plants that were one week old when provided to us, we simply injected Ustilago maydis into the stem of the plant until the solution began to flow from the whorl of the plant. I infected two plants, and I was able to inject about .5 mL spore solution per plant.

Here is an example of an injection of Ustilago maydis into a one-week-old corn plant stem.

There were also two-week-old plants that had been infected one week prior for our viewing pleasure. You can already see galls forming on several of the leaves.