2017 OUTPACE Started!

By Jun Hi Chang

OUTPACE 2017 kicked off with a basic overview lecture of plant pathogens covering:

  • A brief history of plant pathology
  • The interactions that result in plant disease
  • Strategies of plant pathogenicity (how the invaders attack the plant)
  • Different types of pathogens

We first learned that the modern study of plant pathology was kicked off as a result of a terrible tragedy – the Irish Potato Famine of 1845. When a previously unobserved bacteria Phytophthora infestans devastated the genetically very similar (and thus possessing no genetic immunity) potato crops and led to 25% of Ireland’s population being killed, many talented people began studying the potential causes and cures for the potato blight.

One such man was Miles Joseph Berkeley, who in 1846 noticed the sickly black mold on the potato plants, and surmised that the mold was the causality agent of the disease, rather than an effect. In 1863 Anton de Bary took this idea a step further and showed that the bacteria Phytophthora was the cause of potato blight: De Bary transferred spores of the sickly mold from a diseased plant to a healthy plant, and observed that the healthy plant developed the same disease as the original sickly plant. He also directly observed the spores landing and attacking the host plant on a microscopic scale.

The rest of plant pathology research in the 1800s centered on identifying other causal agents of plant disease: experiments in the 1850s demonstrated nematodes as potential harmful parasites, T. J. Burrill and others showed that bacteria were other chief plant pathogens, and finally, Dmitry Ivanovsky identified a virus as the infectious agent in tobacco mosaic. By the end of the 19th century, humanity had a solid understanding of the causes of many plant diseases, and could turn its attention to the prevention of these diseases in the 20th century.

After understanding the history of plant pathology, we learned about the three basic interactions necessary for a pathogen to successfully cause disease in a plant host. Three variables are involved in any pathogen attack: the pathogen itself, the host, and the environment. For a pathogen to succeed in its attack and cause infection, the pathogen must first overcome the host plant’s natural defenses, the host plant must be susceptible to the pathogen, and the environmental conditions in which the attack occurs must be favorable for the pathogen while being disfavored to the defender. Pathogens can increase the chance of a successful attack by possessing favorable genes for evasion, survival, and propagation, as well as simply being great in number. Potential pitfalls that make a host more vulnerable include being in initial poor health and possessing few to no disease resistant genes (such as the population of potatoes in Ireland in 1845).  Environmental agents such as temperature, precipitation, nutrient content of soil, and other organisms’ presence may tip the balance in either way.

Next followed a discussion on strategies of pathogenicy: what must a pathogen do to actually attack into the host? Dr. Mukhtar’s Lecture 1 ppt. page 17 gives us the following requirements:

  • Find the host and attach to it physically
  • Gain entry through plant’s impermeable defenses
  • Avoid the plant’s defense responses
  • Grow and reproduce
  • Spread to other plants (or other parts of the same plant organism_

A chief part to the plant pathogen’s plan is getting past the physical barrier of the plant: how will the attacker actually access the target invasion location? Fungal pathogens use the appressorium to pierce the plant cell wall or use chemical warfare (enzymes) to digest the wall through to gain access. Other bacterial pathogens may use preexisting openings in a plant host (such as the stomata or hydathodes) to enter the plant without much resistance.

Understanding how the invasion process works, the final point of the first lecture was on the three categories that pathogens may fit in: biotropes, necrotropes, and hemibiotropes. Necrotropic pathogens immediately kill the cells that they attack and consume the nutrients from the dead cell carcasses, while biotropes keep the host cells alive and suck nutrients from its host. Hemibiotrophs have the ability to switch between biotrope and necrotrope mode – they can pretend to be in harmony with the host while being ready to flip the kill switch should the moment arise.

The second part of the kickoff week for OUTPACE 2017 consisted of review (or learning) some basic microbiology techniques. The first lab consisted of learning how to propagate bacteria and isolate single colonies. The primary technique involved in this exercise was bacterial streaking, a simple “diffusion” process to spread thin a collection of bacteria to form a location where single colonies of the bacteria can be harvested. Streaking techniques were performed in two agar plates rich in nutrients as well as two tilted agar test tubes. A simple liquid medium for bacteria propagation was also obtained.

For the two agar plates, T-Streak technique was used. The necessary gear for the T-Streak was a preexisting culture of the target bacteria, an inoculation loop, the medium in which the bacterial will be propagated (the agar plates), and a lit Bunsen burner for sterilization.

The process for the T-Streak is as follows:

  1. Purify the inoculation loop by passing it through flame.
    1. Let the loop cool down 20 seconds after it exist the flame. Do this every time you sterilize the loop.
  2. Dip the loop in the preexisting culture of bacteria.
  3. Take the loop full of bacteria and touch the tip of an agar plate (on the circumference of the circle). Make a zigzag motion perpendicular to the radius line from the tip of the circumference as to cover about 20% of the whole agar plate with the bacteria.
  4. Sterilize the inoculation loop through fire again as to remove all bacteria from it
    1. Again, cool down 20 seconds.
  5. Go to a corner of the first bacterial spread zone on the agar plate. Using approximately 25% of the surface area of the first streak, make a zigzag motion perpendicular to the original streaking as to cover another 20% of the whole agar plate with a small percentage of the original streak’s bacteria. This procedure “diffuses” bacteria concentration.
  6. Sterilize one more time.
    1. Cool down one more time.
  7. Go to a corner of the second bacterial spread zone on the agar plate. Using approximately 25% of the surface area of the second streak, make a zigzag motion perpendicular to the second streaking as to cover another 20% of the whole agar plate with a small percentage of the second streak’s bacteria. This procedure “diffuses” bacteria concentration even more.
  8. Yet again, sterilize.
    1. Yet again, cool down.
  9. Go to a corner of the third bacterial zone and make a zigzag motion perpendicular to the previous zone’s markings, but this time, don’t take your inoculation tool off until you cover the rest of the unmarked area on the agar plate. This process will allow you to find single colonies growing in this particular zone after incubation.
  10. Depending on what bacteria you wish to culture, incubate the agar plate in the correct temperature/condition necessary for bacteria propagation.

Figure 1. The Inocuation Loop.Figure 1. The inoculation loop.

Figure 2. The Inocuation loop being sanitized by fire.Figure 2. The Inoculation loop being sanitized by fire. This was done repeatedly during the experiment to eliminate any leftover bacteria as well as to sanitize the loop.

A simplified process was recorded for the tilted agar test tubes. For one of the agar test tubes, the purified-then-bacteria-contaminated inoculation tool was placed at the bottom of the test tube and dragged around in a zigzag motion as to spread the entire test tube’s surface area with bacteria. For the other test tube, the inoculation tool was placed at the bottom of the test tube and dragged straight up in one linear motion.

A final process was performed for the liquid medium, where the pufiried-then-bacteria-contaminated tool was simply sloshed around in the liquid medium.

After two days, the resultant bacteria propagation looked like so:

FIgure 3. One of the T-Streaked agar plates.Figure 3. One of the T-Streaked agar plates. The student may have had too much bacteria on the loop for the first part, since the first, second, and third zigzag swipes look almost identical.

Figure 4. The zigzag-streaked test tube.Figure 4. The zigzag-streaked test tube. Notice that the entire surface area of the agar is taken up by the bacteria.

Figure 5. The straight-streaked test tube.;Figure 5. The straight-streaked test tube. The straight-streaking is very apparent.

 

Figure 6. The Liquid MediumFigure 6. The liquid medium with the bacteria supercolony free floating.

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