DNA analysis of Colletotrichum sublineolum, a

fungus that causes anthracnose in sorghumNicolas Velazquez, Dr. Clint Magill

Bioenvironmental Sciences, Department of Plant Pathology and Microbiology, Texas A&M University

Abstract Results

Introduction and Objectives

References

Relationship to Career Goals

Methods

Results for growing Colletotrichum sublineolum and Peronosclerospora sorghi in pure culture were successful as there was

significant growth. After the process of DNA extractions, half of the DNA showed high concentrations while the other half showed

low concentrations. This is not an unusual event. The DNA concentrations using the nanodrop are presented in Figure 8 and Figure

9.These graphs show steep curves, meaning that a good sample of DNA was extracted. These high concentration samples were

taken into the next step of amplification. The low concentration samples were discarded. After these samples were ran through gel

electrophoresis and examined under UV light, results showed little amplification. Only lane 1 and 6 showed separation in band

sizes. You can see these results in Figure 10. Even though there was little sign of amplification, it was a successful result. It

showed that the primers were non-functional and was a test that needed to be done.

This experience is relevant to the goal of being a scientist and working in a

lab. This research challenged the ability to create original ideas, test

hypothesis and make observations on findings. These skills are required to

pursue a science related career. It also provided experience using

machines such as the PCR, nanodrop and thermocyclers. These are tools

used in scientific labs and is important to have knowledge on how to use

them.

AcknowledgementsI would like to thank Ezekial Ahn for mentoring and giving me lab assistance. I also thank Dr. Magill for accommodating me for research in

his lab. Sponsors for high impact experiences for BESC and the BESC poster symposium include the Department of Plant Pathology and

Microbiology, the College of Agriculture and Life Sciences, the Office of the Provost and Executive Vice President for Academic Affairs.

Conclusion・The main components of DNA analysis are isolation,

DNA extraction, DNA amplification and DNA

sequencing

・RAPD primers were non-functional because only 2

lanes showed amplification

・The DNA showed both high and low concentrations

DNA analysis was performed on both types of

pathogens, Colletotrichum sublineolum and

Peronosclerospora sorghi, to determine DNA

differences using RAPD (randomly amplified

polymorphic DNA) primers among the samples. Ten

samples of Colletotrichum sublineolum were collected

to perform DNA analysis using the RADP primers.

DNA extractions from the pathogen mostly showed

DNA with low concentrations, but there were few with

high concentrations. The samples were applied into

the PCR (polymerase chain reaction) machine for

amplification so that gel electrophoresis could be

performed. The older version of the PCR machine

was found to be more efficient because it requires

mineral oil to be added to your samples. This

prevents the loss of liquid for better results.Separation

of the DNA shown under UV light after gel

electrophoresis determined whether the RAPD

primers were functional or not. Nine of the ten RAPD

primers showed to be non-functional showing no

band sizes when performing gel electrophoresis.

Materials for DNA analysis

Figure 3: Yeast Cell lysis, MPC

Protein precipitate reagant, and TE

buffer used for extractions.

Figure 5: Polymerase Chain

Reaction (PCR) machine

Figure 4: Centrifuge machine:

mixes solutions and isolates

DNA from other contents.

Cultures of Colletotrichum sublineolum were grown

on two different agars, oatmeal and potato broth.

After the cultures were grown, DNA was extracted

using yeast cell lysis, MPC protein reagent, TE

buffer and the centrifuge machine shown in Figure

3. DNA extractions are performed to remove DNA

from its origin for isolation and purification (Rice

2017). Concentrations of DNA were measured

using the nanodrop. RAPD primers were added to

the samples for DNA amplification in the PCR

machine shown in Figure 5. PCR is a machine that

takes a single copy of DNA and amplifies it into

thousands of copies of a specific DNA sequence

(Boundless, n.d). After amplification, the samples

were ran through gel electrophoresis for 30

minutes. 10 lanes were used during gel

electrophoresis. Lane 1 through 5 included the T9

RAPD primer along with a positive and negative

control. Lane 6 through 10 included the G11 primer

along with a positive and negative control. Gel

electrophoresis is a technique used to separate

DNA according to their size and to make sure PCR

was done correctly (Khan Academy, n.d). The gel is

then put under ultraviolet light for results on band

sizes.

Sorghum anthracnose and sorghum downy mildew (SDM) are diseases caused by Colletotrichum sublineolum and Peronosclerospora

sorghi, respectively. Anthracnose limits grain production in most areas where it is grown, mainly South America (Tsedaley et al. 2016).

Downy mildew is also a grain limiting disease caused by the oomycete and is mostly found in the coastal bend and southern United States

(Groene 2016). Knowing genetic variation between isolates gives us more information on the pathogen. The objective of this research was

to determine genetic variation of Colletotrichum sublineolum and Peronosclerospora sorghi by using the components of DNA analysis and to

determine if the RAPDs primers were functional. I hypothesized that all ten of the RADP primers would be functional in producing band

sizes to show genetic differences. Since most of the RAPD primers showed no band sizes, it caused a problem in proceeding to the next step of sequencing. Another gap included the time required to cause contamination on agar, which also slowed down the research process.

Figure 10: Band separation

in lane 1 and 6

Binyam Tsedaley, Girma Adugna and Fikre Lemessa, 2016. Distribution and Importance of Sorghum Anthracnose (Colletotrichum

sublineolum) in Southwestern and Western Ethiopia. Plant Pathology Journal, 15: 75-85.

Boundless. "Amplifying DNA: The Polymerase Chain Reaction - Boundless Open Textbook." Boundless. Boundless, 26 May 2016. Web.

03 Apr. 2017.

"Gel electrophoresis." Khan Academy. N.p., n.d. Web. 03 Apr. 2017.

Groene, Grant . "Sorghum Downy Mildew (Field Facts)." Sorghum Downy Mildew | Milo. N.p., n.d. Web. 04 Apr. 2017.

Rice, George. "DNA Extraction." Genomics. Microbial Life, 03 Apr. 2017. Web. 04 Apr. 2017.

Figure 8: DNA concentrations

measured in ng/uL for

Colletotrichum sublineolum

Figure 9: DNA concentrations

measure in ng/uL for

Peronosclerospora sorghi

Figure 6: Gel electrophoresis