Lab 11

                                  Unknown fungi identification

Objectives:

1)    Practice the skills of fungi isolation and purification from different material.

2)    Apply the knowledge we learn from class to identify the fungi we isolate. Familiar with the distinct feature of different fungi.

Material and Methods:

1: Fungi were isolated from the plants such as: aloe, ficus, dianthus, green pepper, and garden water, soil.

2: Olympus compound microscope.  One is available with an attached camera.

           3: A kit containing microscope slides, cover slips, needles, and transfer loops.

          4. Immersion oil and dropper bottles for water to suspend specimens. 

           5. Lens paper for cleaning objectives, and Kim wipes for working with microscopic slides and specimens.

           6: Bunsen burner, dissecting needle, scalpel, scissors, forceps

7: 70% ethanol, autoclave water

8:  Agar plates, ½ PDA plates

Procedures:

 1:  Collected sample from garden.

 2: Cut the region of interested sample, and performed surface sterilized by 70% ethanol for 2 mints.

 3: Placed on agar plates or ½ PDA plates.

            4: Incubated at 28 degree incubator for 1-2 weeks allowed the fungi to grow.

5:  Sub-cultured the different colonies in same plate to new plate, incubated at 28 degree incubator, change light cycle to 12/12.

6: Observed the hyphae and conidiophores at microscopy. Key book was used to identify the fungi we isolated.

Sample collect---> Suface sterilized---->Culture on plates--->Sub-cultured---->Identification

Observations:

Unknown fungi I:

Fig.1. Dianthus growed in garden behind Borlaug building was suffering the pathogen infection which cause the wilt symptoms at the leave and flowers.

Fig.2. Flowers and uper leave were collect as my material to isolate fungi.

Fig.3. Cut a small part of flower and uper leave from infected Dianthus, sterilized with 70% ethanol, and place on 1/2 PDA plates or water agar plates.

Fig. 4. 2 weeks later, after 2 times sub-cultured, I got purify colony, and when growth at 12/12 light peroid chamber, the isolated were became to produce spores.

 Fig. 5.   Short conidiophores, reduced to phialides. Conidia are dark to subhyaline, simple, globose.

 

My isolated                              Phialophora cinerescens                     Phialophora verrucosa

                  Fig. 6. Compare my isolated with Phialophora. The conidiophore is very similar.


Unknown II:

Fig.1. Aloe was fund in garden under very severe pathogen infection.

Fig.2. Infected aloe was collected and took a picture.

Fig.3. After surface-sterilized, the small parts of aloe were placed on 1/2 PDA plate. Incubated at 28 degree chamber with 12/12 light period.

Fig. 4. Several coloies were growed on plates. Each colonies were sub-cultured to new PDA plates. 2 weeks later,

Fig. 5. My isolated. 

Spores are produced along the vegetative filaments by a swelling of the wall and subsequent isolating by a cross-wall. The conidia can be terminal on the filament or at various sites along the length. Chains of the conidia may occur and are sometimes separated by empty cells.

Chrysosporium tropicum showing typical pyriform shapred conidia with truncated bases which may be formed either intercalary, laterally or terminally.The chain of conidia can be separated by empty cell.

Fig.6.  Chrysosporium

Compare the known  Chrysosporium strucutres, it is convicing to say that my isolated is Chrysosporium fungi which belong to Ascomycota.

Unknown III

Fig.1. Ficus tree were found in garden which was suffering very severe pathogen infection

                       Fig. 2. Leaf was collect and cut into small parts for surface-sterilized.

Fig. 3. Sterilized tissues were places on 1/2 PDA plate to culture the fungi.

Fig. 4. After several rounds of sub-cultured, pure colony was isoloated from ficus. The mycirum were growed to cover the whole plate. Perithecium were formed.

Fungal fruiting bodies are identified by erumpent black, single, globose, very circular pycnidia on the base of needles

Fig. 5. Perithecium were crushed to release the ascospores. Under the microscopy.

                                Fig. 6.  Diaplodia isolated from Juniper ( From published paper)

 

Compare the structure of my isolated and the Diaplodia, together with the symptoms I observed from ficus, indicated that the unknown fungi I isolated is Diaplodia, which belong to Ascomycota.

Discussion: The strategy I used in this project is that, isolate the fungi pathogen from plants, then identify them base on the symtoms they caused and the structures of conidiospores. As we known that specific fungi can only infect very specific plants, furthermore, the symptoms it caused are very unique as well. Taking together these evidences with the observation of microscopy, it is relative easy to identify the unknown  fungi.