Unknown III

Unknown Project: Fungal Genus III

Purpose

The purpose of this project was to isolate and identify genera of fungi not listed in Group I of a picture key of common molds available at the following website: http://website.nbm-mnb.ca/mycologywebpages/Moulds/ID_Plate_I.html. 

Materials and Methods 

Potato Dextrose Agar medium
Sterile Petri dishes
Microscope slides
Cover slips
Squeeze bottle with sterile water 
Tweezers 
Scalpel 
70% ethanol 
Sterile water
15% bleach
50-ml beaker (for ethanol)
Bunsen burner 
Metal striker 
Paper towels or bench cover 
Parafilm 
Incubator set at 25 degrees Celsius 
Tabletop shaker
Compound microscope 
Canon PowerShot SD550 digital camera 

A. Fungal isolation and culture conditions 

Using a scalpel, a section of unhealthy tissue was excised from the leaf of an Indian Hawthorn plant (Figure 1) collected November 18, 2012, at Los Cucos Mexican Cafe in College Station, TX. The leaf section was surface sterilized in 15% bleach for 1 minute, triple rinsed with sterile water (2 minutes per rinse) and then dried on a paper towel. A tabletop shaker was used to surface sterilize the leaf section in a Petri dish (great swirling action and you do not have to hold the dish). Alcohol- and flame-sterilized tweezers was used to plate the surface-sterilized and dried leaf section onto full-strength Potato Dextrose Agar (PDA) medium (Figure 2). The plate was sealed with parafilm and incubated at 25 degrees Celsius in the dark for two days. Fungi that grew out of the leaf section were subcultured to new PDA plates on November 20, 2012, and the plates were sealed and incubated as described. Plates were removed from the incubator on November 25, 2012, and then placed on a laboratory bench to subject fungi to ambient temperature and light, and the circadian clock. Plates were observed macroscopically and microscopically (with a compound microscope) daily during both types of incubation. Closer examination of asexual fungal structures that developed (conidiophores and conidia) was done using a squash mount, as described in Lab 2 of my blog. A sterile technique, also described in Lab 2 of my blog, was used during all plating and microscopic observation, which was performed in Dr. Young-Ki Jo's laboratory in the L.F. Peterson Building at Texas A&M University in College Station, TX.

Figure 1

Figure 2

B. Identification of fungus to genus

One fungus isolated from the leaf section (Figure 2) was identified based on culture morphology and production of asexual structures (conidiophores and conidia), with the aid of published print (Figure 3) and online materials (http://www.studiesinmycology.org/content/72/1/1.full.pdf+html).

Figure 3

  Results

Figure 4. A fungal colony emerged from the leaf section within two days of initial plating on PDA.

Figure 5. Macroscopic view of the top of the fungal colony after growing seven days on a new PDA plate. The colony appeared circular, with a fuzzy greenish-gray center and grayish-white margin.

Figure 6. Macroscopic view of the bottom of the fungal colony after growing seven days on a new PDA plate. The center of the colony was a darker greenish-gray and had a faint ringlike configuration of growth.

Figure 7. Conidia viewed using a squash mount and a compound microscope at 40X magnification. The photograph was cropped and enlarged on a computer to show detail. Notice the different, distinctive sizes and shapes.

Discussion 

Based on colony and conidia morphology, the fungal colony isolated from the Indian Hawthorn plant at first appeared to be a species of Cladosporium. However, after consulting a paper at the website listed in Part B of the Material and Methods section above, the fungal colony could also be Cladosporium-like. I examined the figures shown in Bensch et al. 2012 (Studies in Mycology 72: 1-401), specifically Figure 1, Part 2 (Figure 8). Panel A shows the same conidia morphology that I observed in my fungal isolation. Thus, I am identifying my fungal isolation as Cladosporium-like and of the genus Ochrocladosporium.

Figure 8