UNKNOWN FUNGI PROJECT

 

Sclerotinia spp

Introduction

Sclerotinia disease also known as white mould affect a wide range of crops. Losses ranging from negligible to 100% has been reported in literature. Crops that are affected by Sclerotinia spp include bean, eggplant, lettuce, peanut, potato, soybean and sunflower. White mould is a soil borne disease.

 Materials and methods

Voluntary young pumpkin plants with whitish mold on their base stem from the back yard of my apartment were collected. The samples were washed in running water and then cut into small pieces. The small pieces were then divided into two portions. The first porting was plated directly on 1/5 PDA plates and the second portion was immersed in 30% alcohol solution for 1 minute followed by thorough washing using running water and then plated on 1/5 PDA plates. The plates were incubated for 72 hours at 25C. The samples were checked daily for fungal growth. Using a sterilized scarpel hyphal tips of any growing fungi were subcultered into a fresh 1/5 PDA plates. The subcultered samples were then examined using microscopy techniques for any structures that could be used to identify the unknown fungi.

Results

By looking at the symptoms and signs on the plant I had suspected that the unknown pathogen was probably a Sclerotinia spp. Hyphal properties such as the branching and production of sclerotia confirmed my suspicion that the unknown species was a Sclerotinia spp.

                                          White mould like structures on pumpkin seedling stems

                                     White mould like structures on pumpkin seedling stems

 

                                         

Sclerotinia forming sclerotia

                                       Sclerotia spp hypha branching

Sclerotia spp hypha branching

 

 Conclusions

I can conclude that the unknown fungi were probably a Sclerotinia spp based on the signs and symptoms, on the plant, hyphal characteristics and production of sclerotia. However additional identification of structures such as apothecium which I did not observe in this species would have confirmed with no doubt that the unknown species was indeed a Sclerotinia with all certainty.

 

Cervularia spp

 

Introduction

Cuvularia spp., are darkly pigmented fungi with spores (conidia) efficiently adapted for aerial dissemination. It has very distinctive and beautiful spores, which can be easily identified in both spore traps and direct exams. The shape of the spore is so distinct that it's name came from its shape.

Materials and methods

Leaves from an ornamental palm plant with leaf spots were collected from a flower bed near the Plant Pathology greenhouses. The samples were washed in running water and then cut into small pieces. The small pieces were then divided into two portions. The first porting was plated directly on 1/5 PDA plates and the second portion was immersed in 30% alcohol solution for 1 minute followed by thorough washing using running water and then plated on 1/5 PDA plates. The plates were incubated for 72 hours at 25C. The samples were checked daily for fungal growth. Using a sterilized scarpel hyphal tips of any growing fungi were subcultered into a fresh 1/5 PDA plates. The subcultered samples were then examined using microscopy techniques for any structures that could be used to identify the unknown fungi.

 

Results

Microscopically, the spore appears boomerang shaped to more or less spindle shaped and bent. The spores are smooth walled with 3-4 true transverse septa resulting multi celled spores. Macroscopically, colonies on agar media appear black, hairy and expanding.

                                                  Leaf spots on ornamental palm plant

                                           Leaf spots on ornamental palm plant

                                         Curvularia spp on PDA 48 hours after incubation

                                         Curvularia spp on PDA 7 days after incubation

                                                               Cervularia spores

 

Conclusions

I concluded that the isolated fungi from ornamental palm plant was Cervularia spp

 

Colletotrichum spp

Introduction

Colletotrichum is one of the most important plant pathogens worldwide causing the economically important disease anthracnose in a wide range of hosts including cereals, legumes, vegetables, perennial crops and tree fruits. Among these hosts, chilli (Capsicum spp.), an important economic crop worldwide, is severely infected by anthracnose which may cause yield losses of up to 50%. Typical anthracnose symptoms on chilli fruit include sunken necrotic tissues, with concentric rings.

 

Materials and methods

 Chilli fruits were collected from the Department of Horticulture Organic garden on west campus. The fruits exhibited typical anthracnose caused by Colletotrichum spp. The samples were washed in running water and then cut into small pieces. The small pieces were then divided into two portions. The first porting was plated directly on 1/5 PDA plates and the second portion was immersed in 30% alcohol solution for 1 minute followed by thorough washing using running water and then plated on 1/5 PDA plates. The plates were incubated for 72 hours at 25C. The samples were checked daily for fungal growth. The samples immersed in 30% alcohol were had few other fungi growing such that the plates were directly examined using microscopy techniques for any structures that could be used to identify the unknown fungi.

 

Results

Based on the symptoms and signs on the chilli fruit I suspected that the causative pathogen was a Colletotrichum spp. Microscopy analysis resulted in the observation of conidia and conidiogenous cells.

                                         Symptoms on chilli fruit

Symptoms on chilli fruit

 

                                          Colletotrichum spp on  PDA

Colletotrichum spp on PDA

                                         Colletotrichum spp conidia

Colletotrichum spp conidia

 

 

Conclusions

Based on the above evidence I can safely conclude that the pathogen that caused the symptoms on the chilli fruits was a Colletotrichum capsici.

 

Lab: Isolate pyrethecium from Neurospora crosses, Mushroom Identification and Flourescence Microscropy

Isolate pyrethecium from Neurospora crosses, Mushroom Identification and Flourescence Microscropy


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

C. Flourescence Microscropy
Dr Shaw led us through the various flourescence microscope equipment in his camera lab. To demonstrate the concept we observed hyphal growth of Neurospora and the flourescenct nuclei within cell.

Materials and Methods
An agar block was cut from actively growing Neurospora growing on media. Using flourescent microscopy hyphal tip growth was observed.

Results

A video of Neurospora hyphal tip growth

 

A video of nuclei movement through septa

Mushroom Field Visit

Mushroom  farm field visit

Purpose
The puurpose of the field visit was to observe how  mushrooms are produced.


Introduction
Man  have managed to manipulate the growing environment of edible Basidiomycetes and optimise their production. To this end a wide variety of mushroom are now commercially grown mushrooms.

Materials and Methods
Basically  the mushroom growing process involves the use of substrate such as wheat straw. The straw is prepared by pastuerising it and incubating it through various decomposition stages with the aid of chicken manure and urea under various specific  temperature, oxygen and humidity conditions.
After weeks of  preparing the substrate,  the substrate is packaged in  wooden trays and the substrate is inoculated with spawn in form of grain colonised  with the mushroom fungi. After inoculation, the substrate containging spawn is incuubated at different growing temperatures which are dependent of the mushroom that is being grown until they start emerging and reach a certain size required for harvest.

 Results

                                     Wheat straw before (right) and after(left) decomposition

                                            Decomposed straw in wooden trays

                                      Spawn applied to decomposed straw in incubation room

                Mushroom emerging and masses of mycelium colonising the decomposed straw

                                           Mushroom emerging and ready for harvest

                                          Verticillium problem addressed by aplying salt

                                              Some off type mushroom with exposed gills

                                                              Brown mushroom emerging



Discussion
Mushroom production is one of  the various ways that man appreciates and benefit from fungi, at least it takes away the "bad guy" perception that ordinary people always associate with fungi particularly in their role in both plant and animal diseases.
Interestingly there are also some fungi such as Verticilium spp and moulds that can be problematic in mushroom production. The major challenge for most mushroom producers is mangaging such problematic fungi as they try not  to use fungicides to manage them.


Conclusion
Mushroom production is a well thought way of manipulating growing conditions to optimize the growth of an intended fungi.Challenges still exist especially in managing uninteded fungi and tthere lies opportunities for plant pathologist to develop acceptable management strategies.

Saccharomyces cerevisae: Beer Brewing

Saccharomyces cerevisae: Beer Brewing

Purpose
The purpose of this lab was to demonstrate the role of  Saccharomyces cerevisiae in the process of beer brewing.


Materials and Methods
A protocol from Austin  HomeBrew Supply was used to make  AHS American Amber ale.
Liqiud malt was used as the major starting ingredient. It is important to mantain a sterile environment during the whole process.

 Shan (Left) pouring out the liquid malt extract, Sheila(Centre) stirring the solutin as Danny (Right) watches.

                                                         Dried malt extract

                          Chris pouring out the malt solution and Win wei helps with funnel.

                                                     Testing the gravity of the malt solution

                         Pei Cheng taking a sip of the solution just before final incubation.

                                                   Beeer ready to go!

                                            Adding some sugar before beer botling

                                         Carbonating the beer keg

                                        Finally bottled beer!!

Conclusion
From the looks of things we managed to make un contaminated beer and preliminary organoleptic tests seem to indicate that we got the expected test and alcohol content.

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