Lab 4

Introduction

In lab 4, we observed the different spores produced by Neurospora sp. and then made crosses of Neurospora strains. We were then allowed to observe the interesting dimorphism of Mucor rouxii. Lastly, we observe diseased corn and inoculated healthy corn with Ustilago maydis, otherwise known as corn smut.

Neurospora sp. 
Observations of Neurospora sp. Spores
The first activity of the lab was to observe different spores formed by the Ascomycete fungus, Neurospora sp. This genus makes three different types of spores: microconidia and macroconidia (which can be separated temporally in terms of production), and ascospores. I made squash mounts in order to observe Neurospora SMRP 11 for microconidia. A quick observation of the plate revealed that growth was very sparse (Fig. 1). 

Fig. 1
Sparse growth of Neurospora SMRP-11on SC Mating Media. 

Viewing the slide mounts under the scope revealed that there were plenty of macroconidia, but the microconidia were a bit more difficult to find. Once I did find them, it was obvious that they were structurally different from the macroconidia that were present, as they originated from very small branches and exhibited ruptured phialides from which spores would exit (Fig. 2). 

Fig. 2
Microconidia and conidiophore of Neurospora SMRP-11 at 40x magnification.

I next observed macroconidia from a different culture using both squash and tape mounts. Observing the plate of this culture revealed a vastly different scene than the previous plate, as orange macroconidia wear clearly visible, covering the entire surface of the agar (unfortunately, I did not document this via photography). These conidia were morphologically different from the microconidia, as they looked more like strings of pearls on the conidiophore than short, rectangular branches (Fig. 3, wet mount; Fig. 4, tape mount). 

Fig. 3
Wet-mount of the macroconidia and conidiophore of Neurospora sp at 40x magnification.

Fig. 4
Tape-mount of the macroconidia and conidiophore of Neurospora sp. at 40x magnification.

Next, I obtained the centrifuge tube labeled “X_10021gfpa 9 July à A Female 35 June”, which contained ascospores of a Neurospora sp. I again created a squash mount and observed the spores under the microscope. These spores were large (when compared to the spores seen from the previous cultures), dark in color, and shaped like a football (Fig. 5). It was clear from this activity that the morphological structures of conidia and spores can be variable within a genus, which may depend in part on stresses encountered in the environment.

Fig. 5
Ascospore of Neurospora sp. at 40x magnification.

Neurospora Crosses
After observing the different spores, our goal was to then try to cross different mating types of Neurospora sp. I obtained cultures of SMRP10 (A) and SMRP 11 (a), which were contained within glass vials stopped with a cotton ball, and a sterile SC Mating Media agar plate. I then simply inoculated the left side of the plate with SMRP10 (A) and the right side with SMRP11 (a), then sealed the plate with parafilm (Fig. 6). I will determine if I successfully crossed A and a next week in lab.

Fig. 6
My Neurospora SMRP-10 and SMRP-11 cross.

Observe Mucor rouxii Dimorphism

The second activity of this lab was to observe the dimorphism of Mucor rouxii in a single culture. This species utilizes Oxygen and Carbon Dioxide availability to determine whether yeast cells of hyphae will development. Because of this, there should be more hyphae near the top of the agar, and more yeast cells at the bottom of the agar cup. In order to observed this, I first obtained a small cup of agar inoculated with this species of fungus (Fig. 7).  

Fig. 7
Cup containing agar inoculated with Mucor rouxii. 

I then sliced a thin sheet of agar from top to bottom and placed this on a slide with a coverslip on top. With this technique, I should have been able to see a gradient of hyphal growth transitioning into more yeast cells at the bottom of the cross section. However, this is not what I observed.  I did see sporadic hyphal growth throughout the agar (Fig. 8), but I never saw any yeast cells. I did another slice with this same technique, but saw no observable differences (i.e. density of hyphal growth had not changed, no yeast cells present). I then sliced the entire top surface off of the agar to observe under the scope, and still only saw sporadic hyphae (Fig. 9). The same technique was applied to the bottom surface of the agar, but there were still no yeast cells. Dr. Shaw and Dr. Ebbole were under the impression that these cultures were not old enough yet, and that they may just be exhibiting random fungal remnants which could be an artifact of the lab they were culture in.

Fig. 8
Sporadic hyphal growth of Mucor rouxii throughout agar cup.

Fig. 9
Sporadic and fragmented hyphae near surface of agar cup.

Ustilago maydis
Observe Diseased Corn

The last activity of the lab focused on corn smut (Fig. 10). 

Fig. 10
Un-inoculated corn (left) and previously inoculated corn plants. 

We first observed and documented corn that had been inoculated with Ustilago maydis on September 13, 2012 (Figs. 11, 12), and September 19, 2012 (Figs. 13). I will continue to document the progress of these plants in the coming weeks. 

Fig. 11
Leaf of corn inoculated with Ustilago maydis on September 13, 2012.

Fig. 12
Entire leaf of corn plant inoculated with U. maydis  on September  13, 2012.

Fig. 13
Leaf of corn plant inoculated with U. maydis on September 19, 2012.

Inoculation of Healthy Corn with Ustilago maydis
We then each inoculated our own corn plants with U. maydis. I obtained two plastic containers of corn plants, each of which had two plants. I then injected 0.5 mL of a solution of U. maydis and dH₂O into the shoot of one plant in each container. The second plant was left as a control in each container. I then obtained a container that held a single corn plant mutant which was bred to be particularly vulnerable to U. maydis. I then injected this pant with 0.30 mL of the same U. maydis solution. (Note: I wanted to treat the mutant the same as I had the other plants, e.g. with 0.50 mL of solution. However, this plant was smaller than the others and literally could not hold more than 0.30 mL).  I then labeled all of my plants and placed them at the front of the room (Figs. 14, 15). I will be observing/documenting these plants in the coming weeks as well.

Fig. 14
My three containers holding inoculated and control corn plants. The left and right containers contain two plants each, with only one in each container inoculated with 0.5 mL of U. maydis (the other plant served as a control). The middle plant is a mutant which has been bred to be especially susceptible to U. maydis. This plant was inoculated with 0.3 mL U. maydis.

Fig. 15
Again, here are my three containers holding the inoculated and control corn plants.

Conclusion
It is important to understand the different spores that can be produced by a single species. Neurospora provided the best example of this, with spores that differed in size, pigmentation, and morphology. I feel comfortable with being able to identify these different spore types. As for the Neurospora strain crosses, there should be successful mating at both mating types (A and a) were used. Even though the Mucor rouxii activity did not go as planned, I thought that the ability of a single organism to exhibit such dimorphism at the same time and over a very small distance was amazing. I am sorry that I did not get to actually witness this in class. As for the corn smut, I am excited to see if the inoculations worked!

All for now.