UAB Gardens trip to obtain infected leafs and processing in the lab + Lecture on Phytopathogenic Nematodes by Amir Ahmed

On this warm first day June, the OUTPACE group once again returned to the UAB Gardens. During our previous trip to the gardens about two and a half weeks ago, we found it difficult to find sick plants. The Immunostrip® Pathogen detection kits turned up negative on all leaves tested (See Nicole Lassiter’s “Back to the Garden” for more). However, a couple days before our trip to the gardens there had been a lot of rainfall. Fortunately for us, all the moisture meant that there was a better chance to discover some sick plants.

The garden was full of life

The garden was full of life

We split up into our four lab groups and each group was tasked with searching for samples of infected leaves or fruit from a particular category: Peppers and Eggplant, Beans, Tomatoes, or Cucumbers.

The OUTPACE super sleuths searching for diseased samples

The OUTPACE detectives searching for diseased samples

Finding seemingly infected leaves was much easier this time around. My group was tasked with finding samples of infected peppers and eggplants. Almost immediately, we were able to find leaves that looked suspiciously unhealthy. The leaves were bagged and eventually taken back to the lab for processing.

A suspected diseased eggplant

A suspected diseased eggplant

We returned to lab, eager to test our samples for what potential diseases they could have. First, we performed an experiment to isolate bacterial pathogens. An infected leaf sample was first cut into 4 small squares, rinsed with a 10% Clorox solution then with water, and then put in a grinding tube to grind the tissue.

The sick leaves were taken in for analysis

The sick leaves were taken in for analysis

Ready for grinding!

Ready for grinding!

After grinding, we performed a serial dilution to create 1:10, 1:100, and 1:1000 dilutions using our ground-up infected leaves liquid. We added 5ml from each dilution to a matching label YPD plate. Glass beads were used spread the liquid over the surface of the agar. The plates were taken away to give them time to grow.

Our next experiment was to isolate fungal pathogens. Again, we cut an infected leaf into 4 small squares which we washed with a 10% Clorox solution. We carefully transferred the leaves onto a medium V8 plate. Theses plate were also taken away to be given time to grow.

Turns out fungus love V8 as much as humans do

V8 can help you grow as well as help fungi grow too!

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The next day, the OUTPACE group met again for a lecture on Phytopathogenic Nematodes.

A nematode

A nematode

Beforehand, the first thing that came to my mind when I heard the word “nematode” was a gross wormlike organism that you got if you ate uncooked meat, or those thirsty animals that ate everything and drank Spongebob’s house with straws in that one episode.

Well it turns out nematodes do suck the nutrients out of vegetation, they even use a “straw” of sorts. More specifically, nematodes use a stylet to pierce plant root cells, after which they kill or modify them into nurse cells, and then absorb nutrients from them.

A nematode stylet

A nematode stylet

A nematode stylet unsheathed

A nematode stylet unsheathed

We also learned that nematodes are the most numerous animals on earth. They exist in about all ecological niches and attack almost all plants. A nematode life cycle is 20-30 days and in 1 season a single female can produce 8 billion nematodes.

Life cycle of root knot nematode

Life cycle of root knot nematode

Their cysts are scattered in the dirt, waiting for the right moment to hatch. Nematodes are biotrophs, which means they feed on live tissue, so they don’t set out to immediately kill a plant.

Nematode cyst on soybean root

Nematode cyst on soybean root

However, they can make fungal or bacterial diseases in a plant worsen. Fortunately, plants have a way to fight against nematodes. They have a first layer of defense in the waxy cuticle barrier. Then they have Systemic Induced Resistance, MAMP (microbe associated molecular pattern) and DAMP (damage-associated molecular pattern). Nematodes have their effectors too. One example of a nematode effector is chorismate mutase, which suppresses plant salicylic acid production (a key signaler in defense mechanisms). The gene for chorismate mutase actually came over to nematodes through horizontal gene transfer from bacteria (bacteria have since lost the gene). There are mechanisms of effector-triggered immunity in plants albeit not much research has been done on the subject.

A soybean cyst nematode

A soybean cyst nematode

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