Lab 8

Continuation of Neurospora crassa genetics

Purpose

The objectives of this laboratory exercise on October 17, 2012, were to 1) pick perithecia from Neurospora crassa crosses initiated during Lab 5 on September 26, 2012, and squash them to observe asci and ascospores, and 2) observe field-collected mushrooms.

Materials and Methods 

N. crassa SMRP10 x CSP-1-GFP
Field-collected mushrooms
Microscope slides
Cover slips
Dissecting needle
Dropper bottle with water 
Spray bottle with 70% ethanol
Paper towels
Metal striker
Bunsen burner
Canon PowerShot SD550 digital camera
Olympus SZ30 zoom stereo microscope 
Olympus CX31 compound microscope 

A. N. crassa cross 

While observing a N. crassa SMRP10 x CSP-1-GFP cross under a dissecting microscope, and using a sterile technique (described in my blog for Lab 2 on September 5, 2012), a dissecting needle was used to pick several mature perithecia. The perithecia were placed in a drop of water on a microscope slide. A cover slip was applied and then gently pressed with the handle of the dissecting needle to squash the perithecia and release asci containing ascospores. Fungal structures were observed using a dissecting microscope and a compound microscope that had been set to Köhler according to instructions also received during Lab 2. Photographs were taken through the eyepiece of the microscope using a digital camera. Ascospores with nuclei expressing green fluorescent protein (GFP) also were observed using fluorescence microscopy.

B. Field-collected mushrooms 

A number of field-collected mushrooms that are part of a Department of Plant Pathology and Microbiology collection at Texas A&M University were made available for macroscopic observation. 

Results

A. N. crassa cross

Three-week-old N. crassa SMRP10 x CSP-1-GFP cross used to pick perithecia.

Mature perithecia that were ready to shoot ascospores, as observed using a dissecting microscope (4X). Photograph cropped and enlarged to show detail.

A group of four football-shaped ascospores that were shot from perithecia onto the lid of the culture dish, as observed using a dissecting microscope (4X). Photograph cropped and enlarged to show detail.

Two squashed perithecia released asci containing ascospores, as observed using a compound microscope (10X). Photograph cropped and enlarged to show detail. Each ascus contained eight ascospores.

Using fluorescence microscopy, multiple nuclei expressing GFP could be observed in four of the eight ascospores in this ascus from the N. crassa SMRP10 x CSP-1-GFP cross. Photograph cropped and enlarged to show detail. Nuclei in all eight ascospores should have expressed GFP. It is not known why the nuclei in only half of the ascospores expressed GFP.

B. Field-collected mushrooms 

 

Lab 7

Laboratory practical examination

Knowledge gained during the first six laboratory exercises was put to the test during a laboratory practical on October 10, 2012. Those who took the time to document and understand what was observed and taught during labs should have done well.

Lab 4 update

Ustilago maydis

Using a pair of scissors in my lab supply kit, on October 9, 2012, I excised a leaf from my corn seedling showing symptoms of infection from inoculation with Ustilago maydis on September 26, 2012. I placed the leaf in a Petri dish and covered it with a 2:1 ethanol:acetic acid solution to remove the chlorophyll. Approximately 30 hours later, I drained the 2:1 ethanol:acetic acid solution from the plate and triple rinsed the leaf with non-sterile deionized water. I patted the leaf dry with a paper towel and then covered it with 0.1% trypan blue in lactophenol to stain any fungal structures that may be present. Approximately two hours later I rinsed the leaf with non-sterile deionized water, patted it dry with a paper towel and then mounted it in lactophenol on a microscope slide for viewing using an Olympus CX31 compound microscope. I took photographs through the eyepiece of the microscope using a Canon PowerShot SD550 digital camera. Photographs were cropped and enlarged on a computer to show detail.

Conjugation of sporidia to produce dikaryotic hyphae that are necessary to infect the corn seedling.

Dikaryotic hyphae that appear to be invading the corn seedling via the stomata.

A budding sporidium.

Conjugating sporidia.

Yeast-like sporidia.

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.