OUTPACE 2017 ended with smiles

By Bharat Mishra

It’s always great to have an interactive session on the last day of any course about the overall feedback and forward interests. The environment on June 21 at the OUTPACE 2017 summer research institute was amazing. Time for having a roundtable chat session with snacks and certificates at Department of Biology, UAB.

Figure 1a

 

Figure 1bFigure 1. (a) Discussion table, (b) Mouthwatering Cake.

Session started with opening remarks of course instructor (Dr. Karolina Mukhtar) about OUTPACE 2017 course and participants. She expressed the challenges of multidisciplinary course integration ranging from plants, pathogens (bacteria, fungi, oomycetes, nematodes and insects) and GMO crops to Plant Sustainable Disease Management in a short term course.

 

Figure 2.jpgFigure 2. It’s time for Dr. Karolina Mukhtar to cut the cake.

Afterwards, participants enlisted their views about the overall experience of Lectures, Labs and garden visits. It was most important to note that participants enjoyed the OUTPACE 2017 course content, management and flow of lecture along with microbial lab sessions.

Now, the floor was open to discuss their future interests and challenges. Some (40%) of the participants were very much satisfied with the plant-pathogen interaction as a major research area for their future investigations. While few participants expressed their interest in human medicine, though they were happy about microbiology labs techniques learned during OUTPACE. Dr. Karolina Mukhtar talked about the challenges and competitiveness to get a funded PhD position at UAB. Also, she shared her PhD experience at Max-Planck Institute for Plant Breeding Research, Cologne, Germany.

Finally, Dr. Mukhtar awarded a completion certificate to each participant and discussed the importance and contribution of completing a NSF funded course. A special event came to end with hugs and well wishes to Dr. Mukhtar and participants.

Figure 3.jpgFigure 3. Certificates of 4th Annual OUTPACE Summer Research Institute “Outreach Plant Pathology Clinic and Education” 2017.

Well-deserved and congratulations all OUTPACE 2017 summer research institute participants and team members for successful completion of course!

Figure 4Figure 4. All participants with their OUTPACE 2017 completion certificates.

Genetically Modified Brunch

By Jonathon Carlisle

On Thursday, June 8, we filed into the lab between breakfast and lunch hours as usual, only this time we brought breakfast food samples. The purpose was to test them for genetic engineering. Everyone brought something different, with the result that it was a varied lot. The variety might ensure that some GMO samples would be present, but by accident it also ensured that everyone would be hungry again before lunchtime, irrespective of individual taste. We had rice crispies, apple jacks, and lucky charms, and thats just for cereals. There were also chocolate poptarts and cookies.

Figure 1. some name-brand food items being tested for genetic engineering

All of these delicious food items were broken down and weighed into one gram pieces before being pulverized with a pestle in five mL of water. After this, they were pulverized some more, and introduced to another five ml of water.

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Picture 2. Pulverized Poptart in 10mL water

Once they were finally ground smooth enough for pipetting, we transferred 50 ul each into two separate tubes. One tube was prepared with a plant primer and the other with a GMO primer. The primers “seek-out” a specific region of DNA and amplify it, allowing us to detect the presence of plant DNA in food in one tube and genetically modified DNA in food in the other tube. We kept the tubes in ice-baths to prevent any unwanted or premature reactions. For comparison, we each prepared a known GMO sample, and a known non-GMO sample in different tubes alongside those we prepared from the food brought to lab. We centrifuged them all for five minutes, then put them in the PCR machine.

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Picture 3. tubes chilling in an ice bath.

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Figure 4: a blurry picture of tubes being loaded into the PCR machine.

The PCR machine will heat and cool the tubes in a way that facilitates DNA replication at the sites targeted by the plant or GMO primers present. With just a few cycles, if GMO DNA is in any of the food samples, we will be able to know it.

DAY 2: I made sure to eat breakfast before we returned next week (Tuesday June 13). We transferred the samples by pipette into an electrophoresis apparatus. When a charge is applied to the machine, the negatively charged DNA runs through the gel towards the positive end.  The smaller DNA fragments run further than larger fragments given the same amount of time. By this mechanism the DNA strands are separated based on their size. By already knowing the length of DNA our plant or GMO primers amplify, we can detect their presence by a dark band in the gel at a spot characteristic of certain fragment sizes for a specific amount of run time. The intensity of a band at any spot is correlated with DNA density, and the location of the spot indicates the size of the fragments there.

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Figure 5. a small sample is dropped into the electrophoresis gel.

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Figure 6. Power source.

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Figure 7. Result from gel electrophoresis.

We brought additional food samples this week to test for GMOs. These we tested with simpler field kits, a much faster alternative to the lab technique.

Figure 8. Food samples.

To test these, we placed a small fragment into the mesh provided inside each kit, then mashed it up well and added a test strip. The test strip is dipped in a small sample so that some of the solution slowly creeps upward by capillary action, much like a paper towel dipped in water.

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Figure 9. Ground up sample ready to be tested]

The test strip will show one bar in the presence of a food sample, which means it’s working. If it shows two bars, a GMO is indicated to be present.

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Figure 10. one bar, no GMO detected

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Figure 11: two bars: GMO detected]

Incredibly, the only sample showing two bars was provided by our known GMO control: Jiffy corn muffins.

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Figure 12. Corn muffin mix that is genetically modified.

On the Way to be Pathologists……… Isolation and Culturing of Bacterial and Fungal Phytopathogens

By Taiaba Afrin

On Tuesday, June 6th the outpace team was supposed to go for a field trip to the UAB community gardens to collect infected leaf samples. But it was heavily raining, so our Dr. Karolina and Xiaoyu (our TA) brought some samples for us, it’s very nice of them. We did the lab into two different sections: Isolation and culture of (1) Bacterial and (2) fungal phytopathogens.

(1) Isolation and culture of bacterial phytopathogens: We had our bacterial infected leaf samples. We cut 4 smaller pieces of infected areas and washed them with 10% Clorox solution for 20 seconds to avoid any other contamination other than our desired bacteria and blot on kimwipe. We rinsed the leaf tissues with sterile water and put them into small tubes of sterile water with a grinding beads and grinded them with a homogenizer. We made serial dilution of 1:10, 1:100 and 1:1000 of our bacterial samples with distilled water. We plated our bacterial samples on three different YPD medium plates with glass beads, which was the fun part. Finally, we sealed the plates with paraffin strips and leave them to room temperature to grow the bacteria. After seven days of inoculation we’ve seen the difference for different diluted samples. From 1:1000 plates we have seen the single colonies.

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Figure: 1:10 Bacterial Inoculation plate after 7 days

 

Picture2Figure 2: 1:100 Bacterial Inoculation plate after 7 days

 

Picture3Figure 3: 1:1000 Bacterial Inoculation plate after 7 days

(2) Isolation and culture of Fungal phytopathogens: We had our fungal infected leaf samples. We cut 4 smaller square pieces of infected areas and washed them with 10% Clorox solution for 20 seconds to avoid any other contamination other than our desired fungi and blot on kimwipe to remove excessive Clorox. We plated those smaller pieces into V8 medium plates with sterile forceps to avoid any unwanted contamination. Finally, we sealed the plates with paraffin strips and let them grow for seven days at room temparature. After seven days of inoculation we’ve seen the fungal colonies with their spores on the plates.

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Figure 4: Fungal Inoculation plate after 7 days

This lab was very exciting and effective. I believe everyone was very careful to avoid contamination which an expert pathologist does. We are baby pathologist .

Plant Pathology Students by Day……. Pathology Investigators by Afternoon!

By Ashley Kroeger

Today May 30th, 2017… the UAB Outpace Team took a field trip…. to the UAB Community Gardens! Just located a mere block or two from the UAB’s campus, our team traveled to put to fruition our investigative and prove our skills of identifying plant pathogens. Our research team was split into four groups assigned designated categories of plants. Group 1 observed: Eggplants, Peppers, and Tomatoes/Potatoes, Group 2 observed: squash, cucumbers, and watermelon. Lastly, Group 3 observed bean leaves. Each group was then given packets specific to their category of origin and common pathogens to be observed within each section. The goal of this trip was to observe viral infections of plants and determine the validity of an actual presence of pathogens.

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Figure 1. Photo of UAB Community Garden

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Figure 2. Outpace Pathology Research Student looking for observable signs of infection.

Due to the recent temperament of the weather including frequent raining and ample moisture coupled with sunlight served as a pertinent environment for viral pathogenic growth. From a holistic stand-point the physical attributes associated with: viral, bacterial, and fungal diseases often intersect which proved as a slight curveball for pathogenic leaves. The main attributes associated with specifically viral infections are not limited to: wilting, a yellow discoloration of leaves, and often a mosaic appearance on leaf surface. Some plants had been freshly planted with limited presence of any pathogenic cultivation, while others were extremely engulfed with infection.

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Figure 3. Squash leaves exhibiting the yellowish appearance to surface, curling, and withering.

After proceeding to collect 4-5 leaves of infected suspect viral pathogens the leaves were examined by utilizing  a “ Adgia Pathology Kit”. The kit operates much like that of the exams utilized for pregnancy. A small cross section approximately the size of your pinky finger-nail tip was placed between a liquid solvent and plastic mesh medium. The leaf’s surface area was completely submerged in the solvent, sealed, and grated utilizing a sharpie to press along the outside surface of the mesh. The effect of this mimics grinding leaf tissue utilized for serial dilutions of pathogens. The breaking down of leaf tissue into the solvent allowed for the release of any pathogenic microbes to be brought to surface. After prolonged scraping of leave tissue, an opaque green color was observed in the medium. An immune strip was then submerged into the liquid of extracted leaf tissue. As the strip absorbed the solution, line indicators would be observed to firstly tell validity of test, and secondly verify to correct pathogen to test.

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Figure 4. Technique of leaf extraction.

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Figure 5. Test strip negative results for pathogen.

As previously staged analogy, the test operate as follows. If one red line was observed, the test was often valid for operation. The presence of double lines indicated a valid test, and a positive match of exam to pathogen. Although none of our samples derived a positive match, we did indicate the presence of transgenic crops!

This testing method seems to be observably the fastest for a presence of a pathogen, but you are not always concluded a complete 100% match. The trip was an overall success and I believe I am now more versed to the presence of viral infections within the phenotype exhibited by plants.

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Figure 6. Summer 2017 Outpace Plant Pathology Research Team.

 

Fungi and Oomycetes

By Marissa Brasher

This week in OUTPACE we discusses two distinct types of phytopathogenic attackers, Fungi and Oomycetes. Fungi are the most common plant pathogens with a total of 100,000 fungal species, 300 of these species have been linked to disease in animals while approximately 8,000 species attack plants. Molds and yeasts are widely distributed in the air, dust, fomites, and normal flora aiding the distribution of these infectious agents.  Upon infection, plants appear off-color or yellow, and may be weaker or show signs of wilting. There are three different fungal life styles on plant hosts: biotrophic, hemi-biotrophic/necrotrophic, and endophyte. While biotrophic and hemi-biotrophic/ necrotrophic life styles will eventually kill their host, endophytes live symbiotically with their hosts.

Plants present both primary and secondary defense responses to pathogen infections. Their primary defense is structural, this includes their cuticles, cell walls, suberin layers, and seed coat. Their secondary defense responders are hormonal. Biotrophic plants secrete Salicylic Acid as their defense while Necrotrophs secrete Jasmonates. Fungal pathogens have evolved around these physical barriers through natural openings such as the stomata, wounds, and insect vectors. Some species of fungal pathogens contain a structure called appressoria which is understood to be important the penetration of the plant’s surface. Some appressorias are melanized. Melanized appressoria power penetration by building enormous turgor pressure, the pressure forces the penetration peg through the leaf. The penetration peg grows into a haustorium, which are specialized for nutrient and signal exchange.

Our second lecture this week focuses on Oomycetes. Oomycetes has been previously classified under Fungi, they liken in morphology and physiology but are phylogenetically unrelated. Oomycetes have now been classified in Chromista, there are approximately 500-800 known species. Unlike fungi, their cell walls lack chitin, instead they consist of glucans and cellulose. Their hyphae have no cross walls (septae) and can reproduce asexually or sexually. Asexual reproduction is carried out through zoospores to preserve their genomes. Zoospores have two types of flagella, anteriorly directed is the tinsel type, posteriorly directed is the whiplash type. Sexual reproduction is carried out through a resting spore, an oospore.  Oomycetes are found in water and soil, they can be saprobic on both dead plants and animals. Oomycetes infections can cause downy mildews.

Some are considered deadly plant pathogens. For example, one of the most infamous oomycetes species is Phytophthora infestans, which translates to plant destroyer. This pathogen is responsible for none other than Ireland’s Great Famine of the 1840’s. Phytophthora infestans caused an outbreak in potato late blight, unaware of the pathogen, farmers lost almost all of their potato crops for years causing mass starvation and emigration. Another infamous oomycetes species is Phytophthora ramorum which can cause Foliar Blight or Sudden Oak Death (SOD).  In non-oak hosts the pathogen causes Foliar Blight; however, in oak hosts the pathogen causes Sudden Oak Death. S.O.D. affects mostly true oaks and tanoaks in the United States.

We also conducted a fungal infection on Arabidopsis using the pathogen Botrytis cinerea. Fungal spores were mixed with potato dextrose to later infect the Arabidopsis. We used two strains of Arabidopsis for infection, Col-0, the wild type, and Pad2. Col-0 is more resistant to infection than Pad2. 6 leaves were separated from each strain and taken downstairs for infection. A pipette was then used to deliver 20 microliters of the spore solution on top of the leaves. The leaves were stored downstairs after infection.

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After a few days, we received our infected leaves to quantify the disease severity on the Arabidopsis. Lesions may develop upon infection, leaves may also appear discolored and shriveled. Below is a comparison of the Arabidopsis leaves prior and post infection.

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Before infection                                                      After infection

We expressed the disease severity in the two strains into charts. Below are some photos of the classroom’s data.

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For example, my chart in the middle of the bottom row proves that my Col-0 strain was more resistant than Pad2. The Pad2 strain infection overall produced mostly yellow leaves which shows more disease severity than a light green or gray color. These results conclude our discussions on Fungi and Oomycetes.

The Game is on!

By Kristin Nicole Telford

In this week’s lecture, we learned about the immunity response that occurs in plants in response to a phytopathogenic bacterium. This interaction between plant and pathogen is represented through a zig-zag model. First, a pathogen is recognized by a plant. This recognition triggers Pattern-Triggered immunity (PTI) which then leads to an increased amount of defense for the plant. However, the pathogen has the ability to produce effectors that enhance the virulence of the pathogen by suppressing the plant’s immune response or contributing to the pathogen’s ability to be successful. Once the effectors suppress the defense response of the plant, the plant is now more susceptible to getting sick because its defense response has been lowered. Luckily, some plants have special proteins that allow them to recognize the effector. This recognition will then trigger Effector-Triggered Immunity (ETI) within the plant. ETI acts as the strongest defense response within a plant.

To better understand the process of the immunity response that occurs within plants, we played a game called veggie vaders. This game was played between two people with one person acting as the plant and the other person acting as the pathogen. The person acting as the plant would randomly draw from a stack of cards for plants only, and the person acting as the pathogen would randomly draw from a stack of cards for pathogens only. Because plants have two lines of defense, PTI and ETI, the game was essentially separated into two stages. During the first stage the plant would place down their randomly drawn cards onto the board. Following this, the pathogen would place down their randomly drawn cards. If the pathogen placed down two different cards that directly matched two different plant cards, then the pathogen received points during the first stage. If the pathogen cards did not directly match any of the plant cards, then the plant received points for the first stage. During the second stage the plant placed down another set of randomly drawn cards and then the pathogen placed down another set of randomly drawn cards onto the board. This second stage represented the ETI (effector-triggered immunity stage). If the plant recognized (directly matched) at least one effector placed down by the pathogen, then the plant would win the game through ETI despite what may have occurred during the first stage of the game because ETI is the strongest level of defense for plants. If the plant did not recognize any of the effectors placed down by the pathogen, then the pathogen wins the game. The games played during class were very intense considering some really good candy choices were our prizes and the winner was allowed to choose first. The winner of the game was Travoris Cameron.

Although this game was purely for fun, it did a great job of showing how the plant immunity response really works. There are many effectors produced by pathogens, so it is a game of chance even in the natural environment. If the plant does not have the special protein necessary to produce and effector-triggered response to increase its defense response it is likely that the plant will die.

Pseudomonas Infection

By Emily Stewart

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Healthy arabidopsis plants

This week in lectures we disccussed plants immune response to various pathogens, focusing mainly on bacteria. Pseudomonas syringae is a hemibiotrophic bacteria, so when it infects a plant, the plants follows biorophic immune pathways and will activate the hypersensitive response causing localized cell death. After the bacteria has multiplied to a certain concentration within the plant, it switches to necrotrophic pathways, where the cell death from the biotrophic pathways helps the infection progress.

In the first lab this week, we infected four week old Arabidopsis thaliana plants with Pseudomonas syringae. The technique is important for this procedure so we practiced on used Arabidopsis plants with water before innoculating the plants that were used for the experiment. We used needleless 1ml syringes to force a magnesium chloride solution containing the Pseudomonas syringae (from cultures made the previous week) into the stomata on the underside of the fourth and fifth leaves of the plants. The plants were then covered and put in a growth room for two days.

IMG_3146 (3)                  IMG_3148 (3)

Innoculating Arabidopsis thaliana                      Innoculated plants in the growth room

In the second lab, we obtained samples from the infected leaves and made bacterial cultures from them. To untrained eyes it was hard to identify the infected leaves, so it was fortunate that we had marked them before hand. After homogenizing tissue from each plant and magnesium chloride solution, we made six 10x serial dilutions. We then dropped 20µl of each dilution onto a KB-Strep plate. After incubating for two days, we were able to count individual colonies in some of the dilutions. Using the number of colonies in a spot on the plate, we can approximate how much bacteria is present in the plants.

IMG_3157 (2)      IMG_3158 (2)

Homogenized tissue samples                                               Serial dilutions

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Bacterial culture plate

All of the infections were pretty successful. Most of the infected leaves showed a pseudomonas syringae growth logarithm between 1.E+06 and 1.E+07. Two peoples plants showed slightly less bacterial growth than the others and had a growth logartithm slightly less than 1.E+06 and one person’s plants showed a growth factor a bit higher than 1.E+07.

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Veni Vidi Vici: We learned, we infected, we counted

By Sarah Coffee

We have begun to work with arabidobsis thaliana! But first, we learned about how plant infection works.

There are only eight genera of bacteria that infect plants. In our case, we infected the poor plants with pseudomonas syringae. We learned in lecture how bacteria usually enter through open wounds or natural openings in the plant, such as the stomata. To help the bacteria out, we used flat headed syringes to physically push the serum containing bacteria into the leaf stomata. We chose (and marked) two leaves on each plant that were not too old and not too young, and flipped them over and injected. If injected slowly enough and the right way the leaf will darken as it absorbs the serum. This is a delicate art in itself, so we tried (and failed) with normal water first.

Once they were infected we placed them in the plant room to allow the pseudomonas to grow for a few days. Bacteria thrive in warm and moist conditions. While the bacteria grew, the plant began its defenses, represented with the Zig-Zag Model. It starts with PTI, or Pattern-Triggered Immunity. This is where the plant uses its PRRs (Pattern Recognition Receptors) which are normally leucine rich, repeat receptor kinases. These structures recognize highly conserved elements of the bacterial genome, such as a section of the flagellum that all bacteria have. These PAMPs, or pathogen associated molecular patterns, activate the plant defenses through PTI. However, the bacteria will then release effectors that turn off PTI, resulting in Effector-Triggered Susceptibility. The plant fights back using Effector-Triggered Immunity (ETI), where the plant may recognize some of the pathogens receptors. This game of cat and mouse can go either way, with both parties constantly evolving. Something as simple as environmental conditions may sway the outcome of plant vs pathogen!

The following Thursday we retrieved our plants and began cutting out small circles from the leaves that were infected. (pic1-pic3) Besides having been marked, infected leaves could be determined from the yellowing of the leaves, marking spots where the plant had fought back against the bacterium.

Figure 1-3.

In lecture we learned about biotrophs and necrotrophs which all use different methods of infection. The bacterium we used today, pseudomonas syringae, is a biotroph. This means that the plant will respond with the stress salicylic acid; if it were a necrotroph it would use jasmonic acid. Crosstalk between the hormones prevents both from being activated at the same time; if faced with a choice between the two, the plant will produce Salicylic acid defenses. Salicylic acid triggers the Hypersensitive Response (HR) that results in the production of ROS (among other compounds). The ultimate result is that the plant undergoes necrosis of its infected cells in order to quarantine the bacteria to those few spots; this is what causes the yellow spots we see on the leaf. While this plant looks sick, it is still alive and fighting back.

Once we cut out our pieces we put them in grinding tubes and sent them to the homogenizer. (Pic 4) We then made several dilutions of this bacterial solution and placed these onto agar plates (pic5 and pic6).We diluted so that at some level the pseudomonas would be in individual colonies, making it easier to count with the naked eye.

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Figure 4.

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Figure 5.

pic6Figure 6.

Where did we get these plates? Well we made them ourselves in Lab #2! We split up into four groups with different medium recipes: YPD (modified), V8,YDC (modified), and KB solid medium. After mixing all the ingredients together, we autoclaved the solutions. We then cooled them, stirred them, and voila! They were ready to pour out onto the plates. After about an hour the plates were ready to bag and store for the next lab!

Back to bacteria: the following Tuesday we counted up our colonies. (pic7) Using the dilution number, we figured out how many bacteria had successfully grown inside our plants. All of us had very sick plants!

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Figure 7.

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.

Viruses!

by Katrina Sahawneh

After weeks of working with stubby Arabidopsis plants, it was exciting to walk into the lab and see a table full of tall leafy plants that we soon found out were Tobacco plants, or more formally Nicotiana tabacum.

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Figure 1: Nicotiana tabacum plants waiting to be infected.

Even more exciting, to me at least, was that we would be infecting these plants with Tobacco mosaic virus (TMV). Previously I have done a lot of work with bacteriophage, viruses that infect bacteria, so I loved getting to work with viruses again, but on a different host. Tobacco mosaic virus is also the first virus ever to be discovered and is what first opened the world up to the idea that there were tiny, nonliving particles that could cause diseases.

So what exactly are viruses? I’m sure all of you know the name, but a virus is a nucleic acid (like RNA or DNA) surrounded by a protein coat that is able to organize its own replication only in suitable host cells. There are over 400 plant viruses, and although there are some of every type of virus, they are primarily single stranded RNA viruses. The tobacco mosaic virus is a small, 6400 nucleotide long, single-stranded RNA virus that contains 3 genes, a movement protein, a coat protein, and a replicase. The replicase also contains a leaky stop codon so that sometimes the whole  length is translated, but other times it stops the protein short so that two versions of the replicase protein are produced. Both of these versions, as well as the other two proteins, are necessary for the virus to be able to replicate itself. What really blew my mind was how small this virus’s genome was. Bacteriophage have comparatively large genomes of about 60,000 to 120,000 base pairs, and contain many different genes, many of whose function is unknown.

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Figure 2: Electron Microscope image of the Tobacco Mosaic Virus. It is 18 by 300nm.

In the lab, we got to infect two different Nicotiana tabacum cultivars. One is susceptible, and the virus is able to spread throughout the plant and cause severe symptoms. One is resistant because it carries the N resistance gene that can recognize the replicase protein from TMV. This recognition leads to cell death that can be seen as small necrotic (grayish) lesions a few days after inoculation.

To infect leaves of the tobacco plant, we put a little carborundum powder (black silicon carbide), 20 uL of TMV solution, and then rubbed it gently around to spread it out over the leaf (Figure 3). The carborundum powder was there to make tiny cuts on the leaf’s surface to aid the virus’s passage into the plant cells, but we had to be careful not to be too rough or we would inflict mechanical damage on the plant (figure 4).

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Figure 3: Corborundum powder, the added 20uL of TMV, and it spread around the leaf.

Figure 4: Someone was a little too enthusiastic when spreading the virus and severely damaged the leaf on top. The leaf on the bottom has minimal damage along the veins, as comparison.

Figure 4: Someone was a little too enthusiastic when spreading the virus and severely damaged the leaf on top. The leaf on the bottom has minimal damage along the veins, as comparison.

A week later we got our results back and at first it was a little hard to see the difference between the resistant and susceptible plants. However, upon closer examination, we noticed that the resistant plants had small, gray lesions (Figure 5), while the susceptible ones did not.

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Figure 5: Leaves from the TMV resistant cultivar. ‘Mock’ was infected with water as a control while ‘high’ was infected with 50ng/uL. Notice the gray lesions on ‘high’ caused by hypersensitive response and ignore the light tan mechanical damage.

It was confusing to us as to why the resistant cultivars would be showing clear symptoms, while the susceptible ones were not. Shouldn’t it be the other way around? However, the fact that we could see lesions meant that the resistant cultivars were recognizing the TMV and initiating hypersensitive response, or purposeful cell death, in order to stop the infection from spreading. The susceptible cultivar had not figured out that it was infected yet, and as the infection had not had time to spread system wide throughout the whole plant, there were not many obvious symptoms.  Figure 6 is what tobacco looks like when an infection has “gone viral” and has spread throughout the whole plant, as compared to a healthy plant in Figure 7. We confirmed that the plant was infected with TMV using a field test that was positive (Figure 8).

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Figure 6: Nicotiana tabacum systemically infected with TMV. You can see where the virus gets its name from as the leaves look splotchy and like a mosaic.

 

Figure 3b: healthy Nicotiana tabacum

Figure 7: healthy Nicotiana tabacum

Figure 3c: field test positive for TMV. The first band indicates the test works, and the second indicates the sample has TMV particles.

Figure 8: field test positive for TMV. The first band indicates the test works, and the second indicates the sample has TMV particles.