Microorganisms that are infecting a plant can be identified by sending the diseased plant (or part of it) to a diagnostic laboratory for analysis. The value of identifying the disease causing microbe is to help in determining options for combating the pathogen, as well as monitoring the risk to uninfected plants. It is not uncommon for a farmer to lose a huge portion of their harvest to disease.
There are two major approaches to identifying plant pathogens, and both types are useful and often used together. The traditional, or classic method, relies on observation of the diseased plant and culturing of the pathogen. Based on the disease characteristics, and those of the cultured microorganism, the disease causing microbe, called a causative agent, can be determined. This approach takes time, especially for the causative agent to grow in culture (and not all pathogens do grow in the lab). The second approach relies on genetics and uses molecular techniques to examine the nucleic acids of the pathogen, and classify it based on its genetic sequence.
The sequence of a pathogen’s DNA (or RNA for many viruses) is a molecular fingerprint of that organism. Techniques developed over past several decades now allow scientists to rapidly characterize any organism based on its genetic sequences. This technology is very useful in medicine and agriculture for monitoring and detecting disease causing organisms (as well as anomalies within people, such as cancerous cells). The technology for testing relies on the polymerase chain reactions (PCR) and/or next generation sequencing (NextGen sequencing). PCR may sound familiar as it was extensively used to detect Covid during and since the pandemic.
PCR relies on knowing the genetic sequence of the pathogen. By clever use of purified enzymes and synthetic DNA, targeted sequences of nucleic acids can be detected very rapidly, i.e., a couple of hours or less. NextGen sequencing is a very different approach where all the genetic material, usually in the form of DNA, is sequenced. The data is then compared to a database of previously sequenced organisms, where a match is hopefully found. NextGen sequencing is very thorough in gaining insight to the pathogen, but it takes much longer, and can be expensive.
Both approaches start by isolating nucleic acids. With plants, isolating nucleic acids can be very challenging. This experiment will use a Synergy 2.0 Plant DNA Extraction Kit, with user friendly reagents that yields “clean” DNA. Clean refers to DNA that has been isolated without impurities commonly found in DNA preparations from plants (for more information, click here).
SYNERGY™ Materials
- Synergy™ 2.0 Plant DNA Extraction kit (SYNP 02-100-02)
- Synergy™ Homogenization Tubes
- Plant Homogenization Buffer
- RNase A solution
- Silica Spin Columns (SSC 100-01)
- Bead Beater (any homogenizer that holds 2 ml tubes should work)
- Isopropanol
- 70% Ethanol, stored at -20°C
- TE Buffer, pH 8/ Molecular Biology Grade Water
- Microcentrifuge
- Microfuge tubes, 1.7 ml, snap cap
SYNERGY™ Method
- Place up to 50 mg of plant tissue in Synergy™ homogenization tube, then add 500 µl Plant Homogenization Buffer. Leafy tissue and young roots homogenize well. Seeds require pre-grinding to meal.
- Homogenize via bead beating (approximately 5K oscillations/min) for two minutes. The lysate should appear milky. If the tube contains foam, repeat the bead beating.
- Once the sample is milky, centrifuge for 5 minutes at 15,000 x g, to pellet contaminants and debris. The homogenization matrix will both disrupt the sample and then capture the contaminants. This will be obvious with leaf samples as chlorophyll will be captured by the solid phase. Pelleting the solid phase will yield a clear lysate. A small hydrophobic layer can sometimes be observed on the top of the lysate.
- Transfer the cleared lysate to a new tube and add 5 µl RNase A solution. Incubate for 15 minutes at 37°C. At this point the lysate can be used for PCR but it might require some dilution if the plant has high levels of PCR inhibitors.
- Add 0.7 volumes of isopropanol and incubate at -20°C for 15 minutes.
- Transfer the solution to a silica spin column, with a collection tube and centrifuge at low speed to slowly push the DNA solution through the silica membrane.
- Discard the flow through and place the column back into the collection tube.
- Wash by adding 250 µl of ice cold 70% ethanol and centrifuge at 8000 x g.
- Discard the flow through and repeat the wash.
- Transfer the column to a clean collection tube.
- Elute in 50- 100 µl of molecular biology grade water or TE buffer and centrifuge at 15,000 x g for 1 minute. The DNA can be stored at -20°C until needed.
Quality Control
Following the purification of DNA, it is important to see how much DNA was isolated and its purity. It is not uncommon for an experiment or protocol to fail, thus researchers need to evaluate their progress when protocols are long. checking the quality of the DNA is relatively easy and can be done with a spectrophotometer than measures light absorption in the ultraviolet range (which is a wavelength generally between 190 to 320 nm). For more details, see Testing for Purity.
Using PCR to Detect Pathogens
Isolating DNA from plant tissue is an early step in the diagnostic process. What looks like a simple drop of clear liquid, i.e., what is produced in the final step of the isolation protocol, is actually a highly complex solution. DNA itself is complex, but what is actually in the tube is DNA from the plant, pathogen (if the plant is infected), and any other microorganism, nematode, or insect that was present in the sample. Without the correct tools, finding DNA associated with a pathogen is like finding a needle in a haystack.
To detect a pathogen, you need a significant amount of upfront knowledge about what possible microbes are represented in that tube. What to do with isolated nucleic acids