Lab 4

Microscopic observation and isolation

of zoosporic Oomycota and Chytridiomycota

Purpose

The objectives of this laboratory exercise on September 19, 2012, were to 1) learn about the asexual and sexual life cycles of Phytophthora sp. and Saprolegnia sp. 3) microscopically observe sporangia and zoospores produced by Phytophthora sp. and Saprolegnia sp. 4) microscopically observe zoosporangia and mitospores produced by Allomyces sp. 5) isolate Allomyces sp. from a soil-water solution using hemp seed as bait.

Materials and Methods 

Prepared cultures of various ages of Phytophthora sp. and Saprolegnia sp. 
Hemp seed baited with Allomyces sp.
Microscope slides

Cover slips

Scalpel

Tweezers

Dropper bottle with water

Emerson YpSs agar plus antibiotic media

DS solution

Micro Petri dishes

20 ul pipettor and tips

Spray bottle with 70% ethanol

Permanent marker
Paper towels

Metal striker

Bunsen burner

Canon PowerShot SD550 digital camera

Olympus CX31 compound microscope 

Olympus SZ30 zoom stereo microscope

A. Observation of drawings and/or video clips of the asexual and sexual life cycles of Phytophthora sp. and Saprolegnia sp.

B. Observation of sporangia and zoospores produced by zoosporic Oomycota

Protocols were provided as references for isolation of Phytophthora sp. on V8 and water agar (WA) solid media, and for induction of sporangia and zoospore production by floating fungal plugs in Non-Sterile Soil Extract Solution (NS-SES).

http://fhm.fs.fed.us/sp/sod/misc/culturing_species_phytophthora.pdf 
http://www.apsnet.org/publications/phytopathology/backissues/Documents/1987Articles/Phyto77n10_1475.PDF 

Prepared samples, consisting of fungal plugs from different aged cultures of Phytophthora sp. grown on V8 and WA media, and then floated in NS-SES, were provided for microscopic observation of asexual fungal structures.


Different aged cultures of Saprolegnia sp. produced from baited cucumber seeds were provided for microscopic observation of asexual fungal structures.

C. Observation of zoosporangia and mitospores produced by Allomyces sp.

Protocols were provided for microscopic observation of asexual fungal structures on hemp seed baited for Allomyces sp. prior to the laboratory excercise.

D. Isolation of Allomyces sp. from baited hemp seed

Protocols were provided to isolate Allomyces sp. from hemp seed baited prior to the laboratory exercise by soaking in distilled water to which soil collected from two sites on campus (pecan pit and horticulture garden near the Peterson building) had been added.

http://bama.ua.edu/~nsfpeet/isolation.htm

Whisler, H.C. 1987. On the isolation and culture of water molds: The Blastocladiales and Monoblepharidales. pp. 121-124. In Zoosporic Fungi in Teaching and Research. eds. M.S. Fuller and A. Jaworksi. Athens, Georgia, Southeastern Publishing Corporation. 

Results 

A. Observation of drawings and/or video clips of the asexual and/or sexual life cycles of Phytophthora sp. and Saprolegnia sp. 

http://www.metapathogen.com/phytophthora/

Saprolegnia sp. 

http://www.abdn.ac.uk/ims/staff/details.php?id=p.vanwest

  
B. Observation of sporangia and zoospores produced by zoosporic Oomycota 

P. infestans

Sporangia stained red with acid fuchsin (40X). Photograph cropped and enlarged to show detail. Classmate Sheila McBride did the staining.

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

Here is a link to a video showing release of Phytophthora zoospores from a sporangium. http://www.youtube.com/watch?feature=player_embedded&v=gDT5Pg3_nsM 

Saprolegnia sp.

Baited cucumber seed.

Picture taken at 40X. Photograph computer cropped and enlarged to show detail.

Here is a link to a video showing release of Saprolegnia zoospores from a sporangium. http://www.youtube.com/watch?feature=player_embedded&v=DHNY5xz1hhY

C. Observation of zoosporangia and mitospores produced by Allomyces sp.

 D. Isolation of Allomyces sp. from baited hemp seed

Hyphae on hemp seed infected from soil sample suspended in sterile deionized water. Soil sample was from the horticulture garden near the Peterson Building on campus. Photo taken at 4X and then computer cropped and enlarged to show detail.

Allomyces sp. ??????????

Allomyces sp. ??????????

Hyphae on hemp seed infected from soil sample suspended in sterile deionized water. Soil sample was from the pecan pit near the Peterson Building on campus. Photo taken at 4X and then computer cropped and enlarged to show detail.

 

Allomyces sp. + contamination?

Is the possible contamination Trichoderma sp.?

Discussion

I did not observe any zoospores in the Phytophthora sp. or Saprolegnia sp. cultures, regardless of age of culture, or mitospores from the hemp seed baited with Allomyces sp., so I posted pictures and videos from the internet on what should have been seen. I believe I isolated Allomyces from baited hemp seed in both the pecan pit and horticulture garden samples that were provided. I am in the process of subculturing those isolates to obtain pure cultures. I had a problem with bacterial contamination on the initial isolations due to a less than sterile technique that had to be employed. I then will post photographs of fungal structures once I am certain the pure cultures are Allomyces.