Lab 6

A survey of mitosporic fungi

Purpose

The purpose of this laboratory exercise on October 3, 2012, was to practice microscopic techniques to examine conidiating fungi.

Materials and Methods

Prepared cultures of various Ascomycetes and Basidiomycetes
Microscope slides
Cover slips
Scalpel
Dropper bottle with water
Spray bottle with 70% ethanol
Paper towels
Metal striker
Bunsen burner 
Canon PowerShot SD550 digital camera
Olympus CX31 compound microscope

Using a sterile technique (described in the report from the laboratory on September 5, 2012, that is posted in my blog), a scalpel was used to cut ~1-2 mm square plugs from prepared cultures of Monilinia fructicola, Trichoderma viride, Colletotrichum coccodes, Botrytis cinerea, Curvularia sp., Epicoccum sp., Alternaria brassicicola, Pestalotia sp., Aspergillus niger, Nigrospora sp., Thielaviopsis basicola, Rhizoctonia solani and Fusarium graminearum. Each plug was placed into a drop of water on a microscope slide, and then each plug was covered with a cover slip and viewed at 4X, 10X and 40X magnification using an Olympus CX31 compound microscope that had been set to Köhler according to instructions also received during Lab 2. Photographs of fungal hyphae and conidia were taken through the eyepiece of the microscope using a Canon PowerShot SD550 digital camera. Photographs also were taken of each plated culture. 

Results

M. fructicola

Lots of white aerial hyphae.

Hyphae are light brown on bottom of culture.

Oval or lemon-shaped conidia (40X). Photograph cropped and enlarged to show detail.

Chains of oval or lemon-shaped conidia (40X). Photograph cropped and enlarged to show detail.

 T. viride

Very distinctive blackish green radial pattern of growth.

Bottom of culture is a light olive green color.

Branched conidophores bear phialides singly or in groups. Small, hyaline single-celled conidia (40X). Photograph cropped and enlarged to show detail.

Chlamydospores (40X). Photograph cropped and enlarged to show detail.

C. coccodes

Distinctive salmon-colored concentric ring pattern. Plate contaminated in center.

Nice color and pattern on bottom of culture.

Elongated hyaline conidia with pointed to rounded ends (40X). Conidia are produced from phialides. Photograph cropped and enlarged to show detail.

Elongated, hyaline, pointed to rounded end conidia (40X). Photograph cropped and enlarged to show detail.

 B. cinerea

Lots of greenish grey aerial hyphae.

Abundant hyaline conidia borne on grey, tree-like branching conidiophores (40X). Photograph cropped and enlarged to show detail.

 Curvularia sp.

Very flat, black radial pattern. Not much aerial hyphae.

Brown, slightly curved poroconidia have an expanded third cell from the pore end of the conidium (40X). Photograph cropped and enlarged to show detail.

 Epicoccum sp.

Black fuzzy colony forms concentric rings and a strong yellow to orange-brown diffusable pigment that discolored the medium.

Multicelled, darkly pigmented conidia are globose pyriform with a funnel-shaped base and and broad attachment scar (40X). Photograph cropped and enlarged to show detail.

 A. brassicicola

Very distinctive, uniform, black concentric ring pattern.

Darkly pigmented muriform conidia (40X). Photograph cropped and enlarged to show detail.

 Pestalotia sp.

Very distinctive brownish black radial pattern with lots of aerial hyphae in the center.

Nice radial pattern on bottom of culture.

Four- to five-celled conidia, with the two or three central cells dark brown, and with two or more apical appendages (40X). Photograph cropped and enlarged to show detail.

  A. niger

Patchy black colony.

Globose, dark brown, biseriate conidial head atop a smooth-walled, hyaline conidiophore (40X). Conidia are globose to subglobose, dark brown to black and rough walled. Photograph taken during a previous laboratory excercise (see Lab 2 report in my blog) and enlarged to show detail.

 Nigrospora sp.

Culture likely contaminated.

Not conidia of Nigrospora sp. (40X). Photograph cropped and enlarged to show detail.

These are the correct conidia produced by Nigrospora sp. Conidiogenous cells on hyaline or slightly pigmented conidiophores are inflated, swollen and ampulliform in shape, and bear a single conidium at the apex. Single-celled conidia are black, smooth and slightly flattened horizontally, and have a thin equatorial germ slit. http://www.emlab.com/s/sampling/env-report-04-2006.html

T. basicola

Distinctive blackish, circular, radial colony with notched edges.

Two types of conidia: darkly pigmented multicelled aleuriospores that arise directly from hyphae, and single-celled, thin-walled, hyaline endospores that arise from the tip of a phialide (40X). Photograph cropped and enlarged to show detail.

Enlargement of lower right corner of above photograph.

 R. solani

Dark brown, flat, radial growth with no aerial hyphae.

This species does not produce conidia and is identifiable through distinct mycelial characteristics, which include wide multinucleate hyphae that branch at right angles, and a slight constriction and septum near each hyphal junction. Mycelia are colorless when young but turn yellowish, or light or dark brown with age.

F. graminearum

Lots of white and light brown fuzzy aerial hyphae.

Nice reddish brown, non-uniform, concentric ring pattern.

Conidia in reddish gelatinous matrix (4X). Photograph cropped and enlarged to show detail.

Multicelled crescent-shaped conidia (40X). Photograph cropped and enlarged to show detail.

 Discussion

All of the fungal species examined were Ascomycetes except for R. solani, which is a Basidiomycete. R. solani also was the only species examined that does not produce conidia. Both the C. coccodes and Nigrospora sp. cultures were contaminated. C. coccodes was contaminated in the center of the plate with a light green colored fungus, which may have been a species of Aspergillus or Penicillium. I did not attempt to find out, but I did see smaller, ovoid conidia amongst the C. coccodes conidia. The conidia I observed from the Nigrospora sp. culture also were not correct. Again, I did not attempt to identify the conidia, which appeared to be binucleate. Thus, I found a photograph from the internet, showing correct conidia, to post in this blog.

It goes without saying that morphology of cultures and fungal structures among the Ascomycetes is varied and sometimes unique. Such variability can be diagnostic. Species of fungi observed during this laboratory exercise were easier to distinguish macroscopically and microscopically than the Aspergillus species observed during the laboratory exercise on September 12, 2012 (see Lab 3 report in my blog). We learned during laboratory exercise that fungi need cycles of light and dark to sporulate. Fungal growth that creates concentric rings in solid media, such as that observed for C. coccodes, Epicoccum sp., A. brassicicola, Pestalotia sp. and F. graminearum, may be due to a circadian rhythm.