Unknowns Project

This is still under construction!

One of our goals for this semester was to isolate and identify 3 unknown fungi (unknown to us, anyway). There is a list of common fungi that we cannot turn in, but we can collect from pretty much anywhere for this project. I work with endophytic entomopathogenic fungi in cotton targeting thrips and spider mites, and so I was curious if these arthropods harbor any fungi in heathy conditions that I should know about when doing work like this. I was also curious if they share similar fungi as the food they feed on. In this case, my colonies feed on excised bean leaves, so I did a comparison of endophytes in freshly cut bean leaves and leaves that had been fed upon for one week. The specific groups that I plated out to look for fungi were:

1. Surface sterilized adult spider mite females
2. Non surface sterilized adult spider mite females
3. Surface sterilized adult female thrips
4. Non surface sterilized adult female thrips
5. Surface sterilized immature thrips
6. Non surface sterilized immature thrips
7. Freshly cut surface sterilized bean leaves
8. One week old surface sterilized bean leaves

Adult female Western Flower Thrips.

Two-spotted spider mite adult male. 

To surface sterilize the spider mites and thrips I placed 10 individuals in 70% ethanol for 1 minute, removed them and placed them into 2% bleach for 1 minute, and then removed them into a sterile water wash. The sterilized individuals were then placed evenly throughout a water agar plate. Each group was plated separately, i.e. thrips were not plated with spider mites.

Surface sterilization of plant material followed the same order and dilution, however, leaves remained in ethanol for 2 minutes and in bleach for 3 minutes. Leaves were placed directly on a water agar plate, and with a sterile scalpel, cut into sections to expose more internal surface area to the agar.

The non sterilized mites and thrips were simply pulled from the colony with a sterile tool and placed on a clean agar plate. All of this work was done under a sterile laminar flow hood on September 27, 2012. Once plated, the petri dishes were sealed with parafilm and placed at room temperature near a window to simulate a natural photoperiod.

The laminar flow hood where I did my work, along with surface sterilization materials, a thrips colony and a fresh bean plant.  

Adult female thrips in a beaker containing 70% ethanol. 

A series of beakers for surface sterilizations. From back to front: 70% ethanol, 2% bleach, sterile water.

Adult female thrips across a water agar plate.

A "condo" containing a thrips colony that has been feeding for one week.

Previously fed upon leaves undergoing their surface sterilization.

Adult female spider mites on their water agar plate. I promise they are there, you just have to look closely.

Fresh bean leaves on water agar that were cut to expose internal leaf tissue to the agar.

After six days, the only plates that had growth were the non sterilized adult female mites, the non sterilized female thrips, and the one-week-old bean leaves. 

From the plates that showed growth, agar plugs were removed from areas with clean boundaries that did not visibly have contamination. They were transfered under the flame of a bunsen burner to new, clean 1/2 PDA plates.

A spider mite showing fungal growth.

What appears to be a thrips egg showing fungal growth.

At one week on the 1/2 PDA, there appears to be three distinct fungi growing in my plates. The used leaf and spider mite palates each have a unique fungus, but the thrips plate appears to have a fungus that has also shown up on the spider mite plate. When I get time, I will put these on slides under the microscope to identify what fungi I might have. I have a feeling that they may be on the common list, so I better get to finding new sources of fungus!

Fungi growing from non sterilized thrips.

Two types of fungi from non sterilized spider mites.

Previously fed upon bean leaf on 1/2 PDA growing an unknown fungus.