Lab: Neurospora Genetics and Mushroom Identification

Objectives    

A. Isolate pyrethecium from Neurospora crosses

Materials and Methods

Crosses on Neurospora species from the previous lab were provided for isolation/picking of pyrethecium.
Mature pyrethecium was identified by virtue of its darker coloration relatively to immature pyrethecium.
Originally, the intention was to isolate mature and dark in color pyretthecium the gently squash it so that it realeases Ascospores.
Due to the fact that most pyrethecium was not read and mature, ascospore picking was then postponed for an additional week.
After a week, ascoscpores were isolated from petri dish lids.
The isolated ascospores were then placed on a agar block from which they were individually picked and put a media slunt.


Results
Immature pyrethecium squash resulted on no ascospores observed. Squashing mature pyrethecium resulted in the relaease of ascospores. At first picking of ascospores was very difficult to achieve however with more practice it became easier to isolate ascorespores

                               Neurospora flourescent nuclei of ascospores ( Picture by Charity)

Discussion

It was  clear that Neorospora pyrethecium with a bic were mature enough to contain ascospores where as the light colored pyrethecium containe  not fully developed ascospores. Neurospora spp crosses resulted from asacopsopres presumably from the respective crosses. Over time the ascopsores could be collcted from peti dish lids mainly because the pyrethecium released them by forcibly ejecting them and hence some collcted on the lid.

Conclusion
Crosses of Neurospora spp resulted in ascospores and picking of Neurospora ascospore is a delicate exercise that requires patience and accuracy isolatina and eventual transfer of ascopsores.

B Mushroonm samples
Objective
Identify Basidia with spores
Finding Bullers Drop
Finding clamp conections in fungal hyphae


Material and Methods
Mushroom specimens were provided for examination using a microscope.
Some small slices of mushroom were mounted on slied fro microscopic examination.

Results

                                                             Ganoderma lucidum

                                                     Laetiporus sulphureus

                                                  Daedaleopsis confragosa

 

                                                 Ganoderma tsugae

                                                 Nidula species

                                                       Nidula species

 

 

                                                  Uknown species

Lab 5: Conidiating Fungi

Conidiating Fungi

Objective

The major objective was to prepare slides of conidiating fungi and observing condiating fungal structures under  a microscope.

Materials and Methods
Fungi samples from various conidiating fungi were taken from plates and used to make slides using the squash method.
Slides were then observed under a microscope

Results

          
                                                               Pestalotia spp conidia

                                                         Alternaria brassicola conidia

Thielaviopsis basicola conidia

 

                                                             Thielaviopsis basicola conidia attached to conidiophores

                                                            Apergillus niger conidiophores

                                                           Epicocum spp conidia

                                               Monilinia fruiticola hyphae

                                                                Fusarium graminearum conidia

Curvularia spp conidia

 

 

                                                            Curvularia spp conidia

                                                     Rhizoctonia solani hyphae

                                                       Colletotrichum cocodes conidia

                                                             Colletotrichum cocodes conidia

Discussion
For most conidiating fungi, it was possible to observe dinstict structures that are associated by that fungi. Such distinct structures can easily be used to identify a a fungi. Caution must be taken to avoid identifying fungi wrongly as some do have stucture which look like from a distant.

Conclusion
Conidiating fungi can be identified on the bases of structures such as conidia , conidiophores etc, however use of a classification keys is importani in confirming their true identity.

Lab 4: Neurospora (Ascomycete) crossing, Mucor (Zygomycete) dimorphism and Ustilago maydis (Basidiomycete)

************ Ustilago component On going************

Neurospora (Ascomycete) crossing, Mucor (Zygomycete) dimorphism  and Ustilago maydis (Basidiomycete)

The objective of this lab were:

i)                    Observation of different structures of Neurospora crasa

ii)                   Crossing Neurospora species invitro

iii)                 Observing dimorpism in Mucor spp.

iv)                 Inoculating corn plants with Ustilago maydis and observe disease progression over time

 Materials and Methods

Neuropsora crasa specimens were viewed under a microscope after preparing slides using the squash slide preparation.

Specimens of Mucor spp that had been plated on media were cut into a thin section presenting the upper layer and lower layer of the media

Crossess of Nerospora were made as follows:

SMRP10 X CSP 1 GFP
SMRP10 X SMRP11
SMRP11 X CSP1GFP

Results

Neurospora crasa

Neurospora crasa macrospores 

 

                                    


Cross results

SMRP10 X CSP 1 GFP

SMRP11 X CSP1GFP

                                                          SMRP10 X SMRP11

                                    

                                       Ustilago maydis on corn at 7 days after inoculation

                                            Ustilago maydis on corn at 7 days after inoculation

                               Stained corn leaf infected withUstilago maydis on corn at 7 days after inoculation

Discussion On going

We were not able to observe the dimorphism of Mucor spp in most samples. It was difficult to identify distinct dimorphisim in the two layers we only yeast form was observed with no hyphae stage observed. This was attributed to the fact that maybe the sample had not been cultured long enough, however when we waited to re examine the Mucor spp after some additinal days of culturing it was contaminated.

 
So far we have noticed gall like structures forming of the corn leaf signaling successful infection of the corn leaf by the pathogen Ustilago maydis.

 

Lab 3: Zoosporic   fungi

Objective

The objective of this experiment was to isolate zoosporic fungi and isolate Allomyces (Chytridiomycota) using a cucumber seed as a bait .

Materials and Methods

The original intention was to zoosporic fungi but we ended up using Oomycetes to observe zoospores.

Water samples containing Phytophthora zoospores were observed under a microscope.

Allomyces were baited from the soil by placing a cucumber seed in the soil solution.

Results

Since the structures we were suppose to observe were elusive, pictures below have been borrowed from cited sources.

Zoospores

  Sporangia and zoospores of P. capsici: (A) sporangia and zoospores; (B) a sporangium releasing zoospores.  Source : Apsnet.org

Allomyces

        source:http://www.buildingthepride.com/faculty/pgdavison/kingdom_fungi.htm

Discussion

Even though we changed samples to Oomycetes, the zoospores were difficult to observe. There seem to be a lower population populations and were scarce in our samples. It is clear from the above pictures of that the zoospores have flagella which propels around.
Allomyces have a meisosporangium which is a thick walled diploid structure that produces haploid zoospores. In addition they do have a mitosporangium where spore formation occurs after mitosis.

Conclusion
With respect to zoospore presence Oomycetes even though they are a fungi have some resemblance with Allomyces whic belong to Chytridiomycota.