Investigation of an Unknown Pyricularia Species on Microstegium vimineum

Kamila Hernandez

Authors:  Hernandez, K., Lane, B., Adhikari, A., Harmon, P.F., Goss, E.M.

Faculty Mentor:  Erica Goss

College:  College of Agricultural and Life Sciences

Abstract

When an invasive species establishes in a new area it can gain a competitive advantage by being released from its natural pathogens. With time, pathogens may emerge on the invasive species and the competitive interactions may change. The Big Oaks National Wildlife Refuge in Madison, Indiana is home to native plant species and the invasive grass Microstegium vimineum. Multiple fungal pathogens have recently emerged on M. vimineum. In the summer of 2018, a new disease was observed on M. vimineum at our Big Oaks National Wildlife Refuge field site. The objective of this research was to identify the pathogen causing this new disease on M. vimineum. Symptomatic leaves exhibited diamond to elongated lesions isolated to the leaf tissue. A Pyricularia-like species was successfully isolated from 13 leaves. Conidia were pyriform with 2-3 cells per conidia. Conidia were15.16-16.97 μm in length (16.24 µm average) and 5.65-8.99 µm in width (7.53 µm average). We sequenced the internal transcribed spacer to identify the isolates. Comparison to sequences in the NCBI GenBank database revealed no matches with greater than 93% identity. Phylogenetic analysis revealed the pathogen as a previously uncharacterized Pyricularia-like species and may represent a new genera. This finding will contribute to ongoing research on the effects of emerging pathogens on invaded plant communities.

Poster Pitch

Click the video below to view the student's poster pitch.

Poster

Click the image to enlarge.
0 0 vote
Presenter Rating
38 Comments
Oldest
Newest Most Voted
Inline Feedbacks
View all comments
Andres Osuna
Andres Osuna (@guest_456)
1 year ago

What are some of the techniques or methods you used to keep everything sterile?

Kamila Hernandez
Kamila Hernandez (@guest_786)
Reply to  Andres Osuna
1 year ago

Sterilization is very important when doing any kind of cell culture. Inside of the incubator I always wore gloves, kept 70% ethanol and a flame handy to ensure that the equipment was sterilized after every use. Even with all of these measures in place, there were lots of times where the cultures would get occupied by bacteria and sometimes even mites. I learned that science is a process of trial, error and lots of patience!

Leyda Marrero-Morales
Leyda Marrero-Morales (@guest_1068)
1 year ago

Hi Kamila, could you please elaborate on your gel electrophoresis and the DNA extraction? Were these results what you expected?

Kamila Hernandez
Kamila Hernandez (@guest_4784)
Reply to  Leyda Marrero-Morales
1 year ago

Hi Leyda,

The Gel Electrophoresis and the DNA extraction were my last steps in identifying the unknown pathogen. DNA was extracted from mycelial tissue using a DNA extracting kit called “Dneasy”. Once we had the DNA we proceeded with gel electrophoresis to amplify the DNA. I made lots of errors while undergoing this process and learned that there are many things that can go wrong within itself. It took several trials until we were finally able to ship the results to Genewiz for Sangar Sequencing.

José Antonio Torres Crespo
José Antonio Torres Crespo (@guest_1088)
1 year ago

Were the results what you expected or was the turnout of your experiments different? What challenges did this bring when analyzing your results?

Kamila Hernandez
Kamila Hernandez (@guest_5306)
Reply to  José Antonio Torres Crespo
1 year ago

Hi José,

After I received my result from Genewiz for Sanger sequencing the database revealed no matches with greater than 93% identity. This is not what we were expecting at all! We had to replicate our experiment several times in order to ensure that we were getting the correct results, since it could have easily been a database or experimental error. However, phylogenetic analysis revealed two distinct groups of previously uncharacterized Pyricularia-like species. After presenting all of our data in maximum-likelihood trees, we came to the conclusion that the pathogen may represent a new genera.

Elizabeth
Elizabeth (@guest_1466)
1 year ago

How is the invasive plant, “microstegium vivimeum,” grown here in Florida?

Kamila Hernandez
Kamila Hernandez (@guest_5856)
Reply to  Elizabeth
1 year ago

Hi Elizabeth,

Microstegium vimineum, otherwise known as Japanese Stiltgrass, is a plant species that is invasive to Big Oaks National Wildlife Refuge in Madison, Indiana. The process of growing it and transporting it to Florida is very tricky, given that we want to ensure that it does not spread into our environment. We have seeds of M. Vivimeum shipped to our campus and we carefully grow the plants under the necessary conditions and inside of a sterile and isolated greenhouse.

Daniel Ocejo
Daniel Ocejo (@guest_1726)
1 year ago

Why did you choose to do this project?

Kamila Hernandez
Kamila Hernandez (@guest_6700)
Reply to  Daniel Ocejo
1 year ago

Hi Daniel, this project was assigned to me by Dr. Erica Goss after telling her about my interests in the field. I worked under PhD candidate Brett Lane from the University of Florida as he guided me through all of the steps. This project came about from our simple curiosity about what exactly was infecting the invasive plant species Microstegium Vivimeum in Big Oaks National Wildlife Refuge in Madison, Indiana.

Gabriel Ocejo
Gabriel Ocejo (@guest_1926)
1 year ago

What will you do going forward with these results?

Kamila Hernandez
Kamila Hernandez (@guest_7220)
Reply to  Gabriel Ocejo
1 year ago

Hi Gabriel,

The process of investigating the unknown species led us to the conclusion that it is a new genera. From here, we will hand the project off to a specialized Microbiologist to do an in depth and extensive classification of the pathogen.

Isabella Zucaro
Isabella Zucaro (@guest_2144)
1 year ago

Did you encounter any failures throughout the process of this research project?

Kamila Hernandez
Kamila Hernandez (@guest_7278)
Reply to  Isabella Zucaro
1 year ago

Hi Isabella,

Yes! We encountered lots of failures. Some having to do with lack of sterilization, unsuitable conditions and chemical errors within DNA extraction kits. We underwent lots of replication and corrected our methodology many times. I did not mind these failures because it helped me in improving my skills and my knowledge on the subject by making me do extensive research in the field.

Maria
Maria (@guest_2240)
1 year ago

Very interesting! I loved learning about this. Could you elaborate on the implications of your results and observations?

Kamila Hernandez
Kamila Hernandez (@guest_7334)
Reply to  Maria
1 year ago

Hi Maria,

Thank you, if you are interested in learning more about it feel free to ask me! As a result of the experiment we concluded that the pathogen is not only a species but a genera. It is important for plant pathologists and agricultural workers to have knowledge on plant pathogens in order to prevent the spread of infectious diseases and possible harm to agricultural crops. For this reason I believe that it is important to investigate not just the effects of this new pathogen but also to answer the underlying question of how and from where this pathogen came to be.

Leslie Rodriguez
Leslie Rodriguez (@guest_2304)
1 year ago

Are there any future experiments you wish to conduct after seeing the results of this experiment?

Kamila Hernandez
Kamila Hernandez (@guest_7380)
Reply to  Leslie Rodriguez
1 year ago

Hi Leslie,
After seeing the results of these experiments I am very eager to find out how this pathogen came to be and for what reason it has infected the invasive plant M. Vimineum. As of now, there are no clear indications of where this pathogen could have come from. However, I find it interesting that it has taken over the niche and wiped out the invasive Japanese Stiltgrass. This makes me wonder about the restorative properties of nature.

Ariana Ortega
Ariana Ortega (@guest_2532)
1 year ago

Given that the plant is native to Indiana, how did you interact with and study it here in Florida? How replicable is your study so that others may test for a similar outcome?

Kamila Hernandez
Kamila Hernandez (@guest_7400)
Reply to  Ariana Ortega
1 year ago

Hi Ariana,

Microstegium vimineum, otherwise known as Japanese Stiltgrass, is a plant species that is invasive to Big Oaks National Wildlife Refuge in Madison, Indiana. The process of growing it and transporting it to Florida is very tricky, given that we want to ensure that it does not spread into our environment. We have seeds of M. Vivimeum shipped to our campus and we carefully grow the plants under the necessary conditions and inside of a sterile and isolated greenhouse. Unfortunately, due to safety concerns replication in Florida would not be ideal without the necessary tools, such as a sterile greenhouse. However in places where Japanese Stiltgrass has grown in the environment this experiment is very much replicable.

Isabella Campbell
Isabella Campbell (@guest_2810)
1 year ago

Great work, Kamila! How did you manage to create an environment similar to the one in Indiana?

Kamila Hernandez
Kamila Hernandez (@guest_7420)
Reply to  Isabella Campbell
1 year ago

Thanks Isabella! The greenhouse where the plant Microstegium Vimineum was grown was kept at 28 degrees Celsius. The plants were also maintained at 100% humidity by frequent watering and we frequently replaced the pots of the plants as the roots expanded. This replicated the conditions similar to the ones in Big Oaks National Wildlife Refuge in Indiana.

Maria Camila Yepes
Maria Camila Yepes (@guest_3004)
1 year ago

How much background in research and biology did you have before starting the project?

Kamila Hernandez
Kamila Hernandez (@guest_7440)
Reply to  Maria Camila Yepes
1 year ago

Hi Camila,
I had an immersed background in Biology, specifically plant biology, before starting the project. However as a first year college student this was my first research project. It was all very new to me and I learned that no amount of previous knowledge will make you 100% prepared to face the real world. You learn as you go! It’s all part of the process and I am still learning from it.

Anays Hernandez
Anays Hernandez (@guest_3658)
1 year ago

Hi Kamila, amazing work! How did you discover the pathogen, giving that it is native to another area? In addition, how was this accurately compared to sequences/pathogens using the database system, where it was discovered that there was nothing like this in that database?

Kamila Hernandez
Kamila Hernandez (@guest_7458)
Reply to  Anays Hernandez
1 year ago

Hi Anays, thank you! Researchers that worked with Dr. Erica Goss came upon it while doing an investigation for a different project. When we turned in our amplified DNA to Genewiz there were no matches with greater than 93% identity, which is highly unlikely. After replicating the process many times we came to the conclusion that it was a new genera, meaning that there is no known pathogen species exactly like it in the world.

Leslie Pendleton
Leslie Pendleton (@guest_3962)
1 year ago

Kamila – just wanted to say great work!! Hope you’re taking care.

Kamila Hernandez
Kamila Hernandez (@guest_7462)
Reply to  Leslie Pendleton
1 year ago

Hi Leslie, thank you. I greatly appreciate it!

Lisa Duran
Lisa Duran (@guest_4564)
1 year ago

Awesome research Kamila! Just had a question. How were you able to see spores growing on your cultures?

Kamila Hernandez
Kamila Hernandez (@guest_7468)
Reply to  Lisa Duran
1 year ago

Hi Lisa, great question. I used different types of microscopes in order to see the macroscopic spores growing. Learning to use SEM and other types of microscopes was a great challenge for me because I had never worked with such equipment before. With lots of practice and help I have been able to learn!

Erica Goss
Erica Goss (@guest_6014)
1 year ago

Excellent explanation Kamila!

Kamila Hernandez
Kamila Hernandez (@guest_7472)
Reply to  Erica Goss
1 year ago

Thank you for all of your support Dr. Goss!

Merari Flores Saldana
Merari Flores Saldana (@guest_6326)
1 year ago

Great job explaining your research! Amazing work.

Kamila Hernandez
Kamila Hernandez (@guest_7478)
Reply to  Merari Flores Saldana
1 year ago

Hi Merari, thanks so much! I greatly appreciate it.

Andres Pulido
Andres Pulido (@guest_7276)
1 year ago

Hi Kamila,
I was wondering what are the consequences you have observed so far of emerging pathogens on invaded plant and the communities they are part of.
Great work!

Kamila Hernandez
Kamila Hernandez (@guest_7504)
Reply to  Andres Pulido
1 year ago

Hi Andres, thank you! It is important for plant pathologists and agricultural workers to have knowledge on plant pathogens in order to prevent the spread of infectious diseases and possible harm to agricultural crops. One widely fast-acting and devastating fungal disease that threatens food safety and security is Magnaporthe oryzae, more commonly known as Wheat Blast. Wheat blast was first found in 1985 in Paraná Brazil and it was responsible for over 75% of yield loss. This underproduction can lead to famine in countries. For this reason it is very important that we have clear knowledge on emerging pathogens and their effects of the plant community.

Allen Wysocki - Associate Dean CALS
Allen Wysocki - Associate Dean CALS (@guest_7578)
1 year ago

Kamila:

Well done. I enjoyed learning more about your research. Your poster was particularly well laid out and easy to follow.

Doc W

Isabella Montoya
Isabella Montoya (@guest_7664)
1 year ago

HI Kamila!

I think you did an excellent job of explaining your research in both your video and poster. I am interested in seeing how your discoveries assist microbiologists studying pathogens in the future. Amazing contribution, well done!