LAB 5

Topic: A survey of mitosporic fungi

Objective: To become more familiar with the microscope and  to gain a general idea about the morphology of mitosporic fungi.

Material:

1.           Cultures of fungi with spores. Various Ascomycetes and Basidiomycetes

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.

Procedures:

1: Slide mounting procedures were the same as previously describe. Use compound microscope to observe the hyphae and conidia.

Observation:

Fig.1 Botrytis cinerea. The conidia looks hyaline borne on grey.

Fig.2 Collectotrichom coccolles. The Conidia are straight, fusiform, attenuated at the ends, 16-22 X 3-4 um.

Fig. 3 Curvularia.sp Conidia are pale brown, with three or more transverse septa (phragmoconidia) and are formed apically through a pore.

Fig. 4 Epicoccum sp Spores are dark brown, globose and muriform (septa in both directions, like a soccer ball)

Fig. 5 Nigrospora.sp.  Globose spores.

Discussion:

The mitosporic fungi are consisted by a large and heterogenous group of fungi whose common characteristic is the absence of a sexual state. Many of the pathogenic fungi in humans and plants belong to this group.

Many crops fungi pathogen are belonged to this group, thus it is very important to diagnose and control them in the field. Used microscope to diagnose is much readily and time saving. So as a plant pathology student, it is necessary to familiar with the morphology of these certain of mitosporic fungi.

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.

Lab 3

Topic: Observation of zoosporic fungi of Chytridiomycota and Oomycota

Objectives: Several groups of fungi produce motile flagellate spores, like Chytridiomycota and Oomycota. However the zoospores differ from group to group. The goal of this experiment is: familiar with the zoosporic fungi and learn how to differentiate chytridiomycota and Oomycota from the structure of zoospores. Furthermore, learn about the life cycle of Phytophthora infestans which include the sexual and asexual life cycles and asexul life cycle of Saprolegnia mixta.

Materials:

1.         Cultures of fungi with spores. Phytophthora infestans and Saprolegnia mixta

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

Procedures:

1:  Agar mount slides were prepared as I descried in last report.

2: The procedures of observation the specimen under the microscope were described last report.

Observation:

Fig.1 Sporangia of P. infestans.

Fig.2 Zoospore of P.infestans.

Fig.3 Zoospore of S.mixta.

Discussions: Although the Chytridiomycota and Oomycota both zoosporic fungi, and all generate zoospores, however the structure of zoospore is quite different between these two fungi. Chytridiomycota produce small zoospores with typically single posterior whiplash flagellum, and Oomycota produce large and kidney-shape zoospores with a posterior whiplash flagellum and an anterior tinsel flagellum. The swim patterns of Chytridiomycota and Oomycota different from each other as well. Chytridiomycota swim with darting and zig-zag pattern, some of the class swim in amoeboid probing pattern, like Spizellomycetales. Oomycota swim like corkscrew-like pattern with random changes of direction, thus base on these features of the zoospores, it is easily to differentiate them under the microscopy.

In this class we also got the chance to isolate the Allomyces sp. from baited hemp seed. Follow the protocol which was provided by Dr. Shaw, I try different medium to culture it. All my culture seems got  badly contamination  of bacteria, I am in the process to subculture it, and hopefully I will get the pure culture.

 

LAB 2

Topic: Methods of visualization of conidiophores for microscopy

Objective:

Learn the Riddell mount method to prepare the specimen which is allowed to observe the intact conidiophores.

Materials:

 1.        Cultures of fungi with spores. Several species of Aspergillus were support in this practice, include Aspergillus oryzae, Aspergillus niger, Aspergillus flavus, and Aspergillus sojae.

 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, Petri dish, bend plastic rod, filter paper.

Procedures:

1: There are two different ways to process Riddell mount.

A: Subculture the fungi on agar bed. As the Fig.1 shown, I directly cut a agar block from the agar plate, placed it on the agar bed, and used the transfer loop to inoculate Aspergillus on the edge of agar block from culture plate.  Gently covered a cover slip on the top of agar block, leave some space to avoid the contact of agar block which allow the hyphae to grow around the cover slip.

B: Subculture the fungi on slide bed.  As the Fig.2  shown, firstly I placed a piece of filter paper underneath the Petri dish, and pre-wet with distill water to keep the Petri dish humid, then a triangle plastic rod was placed on the paper to support a slide and avoid to touch the pre-wet filter paper.  A properly size of agar block was cut from agar plate and placed on the slide, and subculture the fungi on this block as described previously.  A cover slip was used to cover the agar block and allow the hyphae to grow around.

2:  One week later, the cover slip was gently took out and place on the slide for microscope observation.  To avoid accidently squash the specimen and distort the intact conidiophore, some broken cover slips were used to pad up the cover slip which with fungi growth on it.

3: The procedures of observation the specimen under the microscope were described last report.

Observation:

  Fig.1 Agar bed based Riddell mount

  

Fig.2 Slide bed based Riddell mount

    

Fig.3 Aspergillus flavus conidiaphore

 

Fig. 4 Aspergillus paraititis conidiaspore
 

Fig. 5 Aspergillus sojae

 

Fig.6 Aspergillus nidulans conidiaphore

Fig.7 Aspergillus tamari conidiaphore

Discussion:

Riddell mount is a powerful method to observe the intact conidiophores for a lot of fungi. The principle of this method is to mimic the hard and hydrophobic surface of plant surface and promote fungi to form the conidiophores as in nature.  The advantage of Riddell mount, when compare with tape mount or slide mounting, is that the hyphae grow on the surface of the cover slip and form the intact conidiophores

In this class we also have the chance to learn how to choose medium to culture the fungi as different medium have different features. For particular specie and purpose usually need to use the specific medium.  A variety of medium were used to culture fungi in the lab and PDA (potato dextrose agar) was most widely used medium for fungi culture, for different purpose, people used different concentration of PDA. ½ PDA, ¼ PDA were used to culture some particular fungi as they have much limit nutrients.  Because PDA medium is too rich nutrients, so that excessive mycelial growth is obtained at the expense of sporulation. Water agar plate also widely used in lab as fungi and bacteria all grow on this plate but at such slow rate, and target fungi will be much easy to isolate from this kind of medium.