Lab 4 20120926

1. Neurospora crassa (Ascomycete)
Goals: Observe ascospores, microconidia (only produced on poor nutritious agar plate) and macroconidia.
Neurospora crassa is a type of red bread mould and the best-characterized filamentous fungi. It has a relatively simple haploid life cycle. It contributes to biological research by being a model system. It helps Dr. Beadle and Dr.Tatum to raise the "one gene, one enzyme" hypothesis and have been a very useful tool for people to study Genome evolution, circadian rhythm, epigenetics etc.
During the asexual stage of Neurospora crassa, they produce both microconidia and macroconidia. Meanwhile, they produce ascospore as the sexual spores, which unfortunately failed to found by me in the class.

1.1 Macroconidia chain

 Pic 1. Neurospora crassa Macroconidia chain

I can see the intact macroconidia chain by using tape mounts.

1.2 Hyphae

Pic2. Neurospora crassa Hyphae

1.3 Microconidia

Pic 3 Neurospora crassa microconidia (small spores circled with red lines)

It is much easier to find macroconidia than microconidia. The microconidia can be only produced on poor nutritious agar plate.

2. Neurospora crassa mating
For heterthallic Neurospora crassa, they need two mating type spores come into contact, MATA and Mata. In our lab practice, we have smRP10 as MATA strain and smRP11 and NCAC021-1 as Mata strains.

Pic 4 Preparation for mating

2.1 Prepare the strains and medium plates used for mating, and divided the medium plate into half with a Sharpie pen.
2.2 Handle the mating process by the burner and sterilize all the tools like the inoculating needle.
2.3 I picked a little bit spores of SMRP10 and inoculate on one side of the medium plate and then steriled the inoculating needle and picked small amount of spores of SMRP11.
2.4 Sealed with the parafilm, and then put the plate under 25C.
2.5 I also did a control by mating SMRP11 and NCAC021-1, which are 2 MATa strains, and supposed to failure of mating.
2.6 One week later, we observed the mating results. We can see successful mating between SMRP10 and SMRP11, which belong to 2 different mating types. The black dots in the picture below indicates sucess of mating, while no mating product can be observed form the control group. However, the 2 strains didn't mate in the middle of the medium plate perfectly. It may due to spores of one of the strain move faster than the other one.

Pic 5 SMRP10 (A) X SMRP11 (a) successful mating

Pic 6 SMRP11 (a) X NCAC021-1 (a) unsuccessful mating

Pic 7 Perithecia of N. Crassa 

I also observed the black mating product under the dissecting scope.

3. Ustilago (Basidiomycete)
3.1 Observe disease symptom

Pic 8 Disease symptom of Ustilago 1 week after inoculation

3.2 Inoculate the healthy Maize leaves with Ustilago
First, I picked 2 uniform and healthy Maize plants. Then, I used the syringae to inoculate the Maize stem until we can see liquid drop appear around the whirl of the plant. Meanwhile, inoculate another healthy plant with only water.

Pic 9 Healthy plants before inoculation (Left: water, Right: Ustilago)

Pic 10 10 days post inoculation (Left: water, Right: Ustilago)

1 week later, I can see obvious Ustilago tumors on Maize inoculated with Ustilago, while the control plants which only be inoculated with water are still very healthy and green.

3. Mucor Dimorphism (Zygomycete)
When deprived of oxygen, Mucor grows as spherical, multipolar budding yeasts. In the presence of oxygen, they propagate as branching coenocytic hyphae.

Pic 11 Mucor Dimorphism