Lab 4

Topic: Dimorphism, mating and infection of Ascomycetes, Zygomycetes and Basidiomycetes

Objective:

Become familiar with the process of mating with Neurospora cassa different compatible mating type, and observe three different spore type of Neurospora cassa.  Observe the dimorphism of Mucor rouxii.  Learn the life cycle of Ustilago maydis and infect the maize to observe the infection process and symptoms.

Material:

1.           Cultures of fungi with spores. Different mating type of Neurospora as list bellow:

SMRP10 N2281-3 FGSC#9518 mat A ridRIP4 his3+::Pccg-1-1hH1+-sgfp+

           SMRP11 N2524 FGSC#9519 mat a ridRIP4 his3+::Pccg-1-Bml+-sgfp+

            csp-1 mat(???) his3+::Pccg-1-1hH1+-sgfp+ (???)

                Cup culture of Mucor rouxii.

               Two cultivars of maize seedlings, Silver Queen and OPR2-1, and Silver Queen is more susceptible to Ustilago maydis

2.         Olympus compound microscope.  One is available with an attached camera.

 3.        A kit containing microscope slides, cover slips, needles, and transfer loops.

 4.        Immersion oil and dropper bottles for water to suspend specimens. 

 5.        Lens paper for cleaning objectives, and Kim wipes for working with microscopic slides and specimens.

  6:      Bunsen burner, dissecting needle.

  7:      Medium for Neurospora crossing.

Procedures:

A: Observation of different t spore type of Neurospora cassa.  The sample was preparing form medium plate, and mounting method was fellow previously protocol as described before.

B: Crossing the Neurospora cassa with comparable mating type.  The comparable mating type Neurospora cassa were subculture in same plate by transferring the conidia. The fungi will grow toward to each other, there will be a line between when two fungi contact, and the mating will happen in this line, and ascospores may be observed in this area.

C: Observation the dimorphism of Mucor rouxii. Mucor rouxii was cultured in a small agar cup. I used scalpel to cut the agar vertically from the cup, and the thin piece of agar was placed on the slide, further to mount as the specimen to be observed under the microscopy.

D: Inoculation the maize with Ustilago maydis. The spore suspension was prepared before the class, the concentration was adjusted to 10⁵, and we used the syringe to infiltrate the spores into upper part of stem of maize, meanwhile I infiltrated H2O to another seedling as negative control. After inoculation, the seedlings were put back to the growth room under the normal growth condition, one week later, come back to observe the symptom development.

When the leave become to show the gall at the underneath, it is time to stain and observe the fungi in the plant cell. I cut a small part of the characteristic symptom leaf out and further cut into smaller pieces. The leaves were fix and decolorized by 1:2 Acetic acid: Ethanol for overnight, and change to 0.1% trypan blue in  lactophenol for 5 hours.  The leaves were further distained with1:2 Acetic acid: Ethanol for overnight and mounted as specimen to examine under the microscopy.

Observation:

Fig.1:  Microspore of  Neurospora cassa

Fig.2 Macrospore of Neurospora cassa

Fig. 3 Ascospore of Ascomycete

Fig.4 Crossing of Neurospora cassa

Fig.5 Dimorphism of  Mucor rouxii

Fig.6 Inoculation of Maize with Ustilago maydis, the galls were shown in leaves, and hyphae were observed inside the plant cell.

Discussion:

N. crassa is used as a model organism because it is easy to grow and has a haploid life cycle that makes genetic analysis simple since recessive traits will show up in the offspring. Analysis of genetic recombination is facilitated by the ordered arrangement of the products of meiosis in Neurospora ascospores. Its entire genome of seven chromosomes has been sequenced. N. crassa has different mating types determined by the mating genes, termed A and a. It is readily to handle the cross in vitro. In this class, we got the chance to cross two comparable N. crassa and observed that the matting morphology in the plate. But I failed to observe any ascospores although the plate has shown very nice cross line.  May be it will take longer time to generate ascospores or may need a short period hit shock to stimulate the generation. I will update the process in the future.

Dimorphism fungi can exit with different from such as mold, hypha or yeast body. When deprived of oxygen, Mucor rouxii grow as spherical, multipolar budding yeasts. In the presence of oxygen, they propagate as branching coenocytic hyphae. The ease with which these morphologies can be manipulated in the laboratory. In this class we observed that in the topper layer the fungi was exited as coenocytic hypae as the presence of oxygen, and in the bottom layer of the agar, only budding yeast form can be observed due to the lack of oxygen.

Ustilago maydis was belonged to Basidiomycota and causes smut disease on maize and teosinte. Although it can infect any part of the plant, it usually enters the ovaries and replaces the normal kernels of the cobs with large, distorted tumors analogous to mushrooms. These tumors, or "galls", are made up of much-enlarged cells of the infected plant, fungal threads, and blue-black spores. The spores give the cob a burned, scorched appearance. In this class, we inoculated Ustilago maydis on maize seedlings, and successful to observe the galls were formed in the infected seedlings which indicated that the fungus has successfully infected the host. The leaves with galls were examined under the microscopy and the fungi hyphae were observed inside the cell.