As we close on the final week of the 1st Annual OUTPACE Summer Research Institute, I will start by reflecting on what we have accomplished during the past six weeks. During week one, we were introduced to plant pathology and had our first trip to the gardens. We also spent some time reviewing basic microbiological techniques that we have used throughout the remainder of OUTPACE. Week two began with a reflection on our visit to the gardens, along with an introduction into bacteria and the various ways they infect plants. We also did our first experiment, infection of Pseudomonas syringae on Arabidopsis thaliana plants. During week three, we began to learn about fungi, while monitoring the progression of disease from the Pseudomonas syringae infection. At the end of week three, we began our second experiment, inoculation of Alternaria brassicicola on Arabidopsis thaliana plants. For week four we quantified the results from the previous experiment, while also being introduced into phytopathogenic viruses. During week five, students were asked to bring samples of infected plant material to the lab. From there, we prepared plates to grow the various fungi and bacteria. Samples were also cataloged for further use in future OUTPACE experiments. (We had a great time with the liquid nitrogen!) That brings me to the final week of OUTPACE, week six.
Monday July 7, 2014
This week of OUTPACE began with a trip to the gardens to collect samples. We then returned to the lab to plate the samples of bacteria and fungi. We also used ID kits to test for certain viruses.
Tuesday July 8, 2014
A lecture on arthropods was presented in order to give us an introduction into the various relationships that exist among both plants and arthropods. We were given further insight into examples of arthropods that are beneficial to the plant, along with a variety of examples that are harmful. Check out this parasitoid wasp!
The wasp (helpful to the plant) uses a caterpillar (harmful to the plant) as a host in which to lay its eggs. The larvae will feed on the caterpillar until they reach a stage in their life cycle when they are ready to break out. The larvae will then form silk cocoons for protection. This is further aided by the wounded caterpillar, which also forms a web around the larvae as an extra layer of protection. Eventually, the caterpillar will starve to death while protecting the larvae. The larvae are then free to hatch. We all found this to be a pretty interesting relationship. This lecture emphasized the complex relationships among organisms.
Wednesday July 9, 2014
During a previous lecture, the topic of genetically modified organisms (GMO) was brought up. In response to the interest expressed by the OUTPACE participants, we had a discussion about GMOs. For this discussion, each student was instructed to prepare a five minute lecture on this topic, along with their opinion on consumption of GMOs. During the discussion, some interesting perspectives were brought up.
Alison began the discussion with a power-point, giving a thorough background on GMOs. She emphasized the fact that the FDA does not require labeling of GMO products.
Next up was Darion, who also gave a power-point presentation. Darion provided statistics on the top ten crops that are genetically modified. Some of those included corn, soybean, and squash.
Brenna provided information on the potential for horizontal gene transfer among these genetically modified organisms. She also discussed various short term studies that have been performed, while emphasizing the difficulty of tracking long-term effects on GMO food product consumption.
Cody also prepared a power-point to present. Cody talked about the Bt toxin; this toxin forms pores in the intestinal tract of insects. An interesting point that was brought up was the concern that some insects are beneficial to the plant.
For her part of the discussion, Maggie provided insight into the amount of information that we do not know about GMO products.
It is also important to note that GMOs do have positive goals. For example, by creating crops that require less water, this aids countries who do not have adequate rainfall.
Thursday July 10, 2014
Dr. Mukhtar lectured about plant disease management. The lecture started off by presenting the numerous ways that pathogens can spread amongst crops. Wind and rain are two examples. One method of eradication that was discussed was the need to remove infected plant material. When a leaf or portion of the plant is observed to have been infected, it is best to eliminate that portion in order to reduce the ability of the pathogen to spread. Some ways we talked about that would aid in disease prevention were to sterilize the soil before use and use certified seeds. Using seeds that are not certified brings the risk of planting seeds that already carry some sort of pathogen. These measures allow one to take measures to prevent spreading of disease. Another preventative measure mentioned was to plant things that produce odors. This can help prevent insects from attacking the plant. This was seen in the gardens; several gardeners planted marigolds near their plants in order to deter insects.
Another point that was emphasized was that there are pathogens that are known to attack several host plants. So, it is often beneficial to plant these crops at a distance from each other. If these crops are in close proximity, infection of one crop makes it easier for others to be infected by that pathogen. This risk is reduced when they are at a considerable distance from each other. This discussion correlated with what we have been learning from our trips to the gardens.
Friday July 11, 2014
To end the 1st Annual OUTPACE Summer Research Institute, we had a celebration. Each student was given a certificate to recognize their participation. Dr. Mukhtar also shared some excerpts from the “letter to a colleague” students prepared earlier in the week. For this assignment, students were to prepare an email addressed to a colleague to inform them of the OUTPACE program. We had ice cream, brownies, cookies, and fruit to enjoy.
OUTPACE2014 is officially completed! Thanks to all for the fabulous six weeks filled with learning and fun! Please check back next summer to follow the adventures of OUTPACE 2015!
On Monday (June 30, 2014) the OUTPACE began their lab session by inspecting our bacterial and fungal isolations from last Friday. The fungal growth from our samples is progressing as expected and will be further examined on Wednesday. For today’s lab our bacterial isolations must be processed further by collecting single colonies and placing them into a new agar plate by utilizing the quadrant streak method.
Students were also required to bring their own infected plant materials into the lab in order to be tested for certain pathogens. Students were asked to give a prediction of about the type of pathogen that they could have and tasked with isolating the pathogen. My partner, Maggie brought in a diseased potato, which we both used for today’s exercise.
Other samples included a mango, a peach, raspberries and a variety of leaves from our backyards.
The students performed the bacterial and/or fungal isolation method (depending on their material) performed last Friday in which four squares of infected tissue were cut out and sterilized in a 10% Clorox solution. The samples were transferred to a small tube containing water and a grinding bead to be grounded into a liquefied solution. Here is us grinding the samples on a grinding robot:
The solution was diluted three times in 1:10, 1:100, and 1:1000 solutions and placed in three specific plates and spread across the agar with the help of small beads. We concluded today’s lab with a small interview with each OUTPACER and their own personal assessment of the program overall.
On Tuesday (July 1, 2014) there was a lecture on phytoparasitic nematodes and their impact on plant species. We learned that a small percentage are parasitic in nature and are biotrophic. Nematodes are able to produce 300-400 eggs and over the period of one season can produce over 8 billion offspring, which is why they are a substantial problem, especially in agriculture. Nematodes are usually found in soil and will make their way to a root in order to absorb nutrients. The major points of the lecture were that they can live inside (endo-parasitic) or outside (endo-parasitic) and use a stylet in order for them to burrow within the root of the host and cause major problems such as premature wilting, stunted growth, and galling or cysting of roots. Some plant defenses have been observed, but due to natural selective factors there is no real distinct prevention of these parasites. More scientific research is needed to address the genetic basis of plant nematode resistance.
On Wednesday (July 2, 2014) the OUTPACE students were back in the lab continuing from our previous work on Monday. The session began with a brief explanation by Dr. Mukhtar explaining the importance of long term preservation and how using this method allows scientist to study strains of pathogens for future use. We used our inoculated bacteria that we prepared Monday for this exercise.
Using pipettes, 1mL of 80% glycerol is directly added into the bacterial tube and mixed. A small cryogen tube was passed out for each student and 1mL of the bacterial glycerol solution was poured into the tube. Once sealed, the tubes are placed into a liquid nitrogen medium where they will instantly freeze and safely preserved. Here is a picture of the cryo-preserved bacterial glycerol stocks in liquid nitrogen.
Our next exercise involved the fungal cultures we prepared on Monday. After three days of growth, the cultures had undergone favorable progress. However, in order to further purify our samples, a small area of fungi was to be transferred into a new agar place. Sterile instruments and new V8 agar plates were passed out to the students. We hope to see our new fungal plates grow over the long weekend.
Lastly, the students were handed back their infected plate samples from home to observe and further process. We took samples from a single colony and transferred it into a new agar plate by using the quadrant streak method.
Although today’s week was shorter due to the July 4th Holiday, we are always learning new material and methods in the lab and future careers. Look forward to week 6 as we continue our work here in OUTPACE.
Tuesday, June 24th
After a relaxing three-day weekend, the OUTPACE team went back to work with a discussion, a lecture on fungi, and observation of the Alternaria inoculations from last Friday.
The previous week, we were given six questions to answer regarding the basics of plant pathology. We started our day off with a discussion of the questions, which emphasized key points such as the plant’s ability to recognize pathogens, as well as the mechanisms that allow a pathogen to evade detection, such as effector production. A few discussion points required further thinking, however, with the integration of our new-found knowledge of plant immunology with previous ideas covered in introductory biology and genetics courses. Examples included the understanding of horizontal gene transfer between microbes, which can result in the evolution of highly resistant strains, and lack of genetic variation in a population, which can make a set of related plants equally susceptible to pathogens and may be harmful economically.
Fungi are quite the interesting pathogens. Unlike bacteria, which infect plants intercellularly, fungi are capable of piercing the plant cell walls and invading the cell interior to obtain nutrients. Fungi are one of the most common phytopathogens and can be biotrophic, hemibiotrophic, or necrotrophic, like Alternaria. Most infectious fungi are saprophytes, which feed on dead organic matter. A benefit of working with a saprophytic necrotrophic species is that it is an obligate parasite that does not necessarily require a host to thrive; this allows for simple isolation from infected matter in a lab setting. Biotrophs, in contrast, cannot reproduce outside of the host and are more host-specific, making them more difficult to observe in media.
The latent spores of fungal pathogens attach to the surface of the plant and penetrate the natural barriers (the cell wall, cuticle, or seed coat), by entering through wounds natural openings, such as the stomata, or through an insect vector. Mechanical force or digestive enzymes may be necessary for effective penetration. Following successful attachment and penetration, the fungi may release feeding structures, or haustoria, through the plant cells; elongate their filamentous hyphae, and then produce conidia, which are asexual spores, ready for reproduction. Whether or not a fungi succeeds in fully invading and reproducing depends on the plant’s immune strength, which is determined by genetics. Non-host plants are more immune to fungi than host plants and often succeed in thwarting the efforts of the invader.
The day concluded with a visit to the growth facility to observe the Alternaria-infected Arabidopsis leaves. Black and brown lesions were visible on the infected leaves after only four days of exposure to the pathogen.
Wednesday, June 25th
Wednesday began with Alternaria observations. The leaves were beginning to yellow, as expected following infection with a necrotrophic pathogen.
Two weeks ago, our trip to the UAB Community Gardens was postponed due to rain, and Wednesday was used as the make-up day.
Off to the Gardens we go!
The six OUTPACE investigators broke into three teams of two. Each of the three teams dedicated itself to one specific plant: tomato, bean, or pepper/eggplant. Our mission was to identify sick plants and, based on their symptoms, determine the possible causative pathogen with the help of a few handouts and atlases. We were also fortunate to meet several of the growers and discuss their concerns regarding plants’ health.
Amidst the illness, however, we did manage to find a few surprises!
…Including a pepper that housed insect eggs!
A few specimens were collected for Friday’s lab activity:
But, like true plant doctors, we brought along ImmunoStrip Kits (purchased from Agdia, Inc.) to make on-the-spot diagnoses.
Each kit comprises a pouch of liquid buffer and a test strip designated for a specific pathogen, such as the Tobacco Mosaic Virus or Clavibacter michiganensis. A sample of the infected plant in question is added to the pouch, and then macerated with the buffer. The specific test strip that corresponds to the suspected pathogen is dipped in the buffer-sample solution.
We waited patiently for the solution to travel up the strip. In some cases, this may take up to half an hour, but some obtained results within ten minutes.
My partner, Audrey, and I collected samples from a tomato plant that we suspected was infected with either Tomato Spot Wilt Virus (TSWV) or the well-known Tobacco Mosaic Virus (TMV.) The leaves of the plant appeared dry, wilted, and curled inward- a symptom that is often observed with either of these two viruses. She obtained a TMV ImmunoStrip, while I chose a TSWV strip, and we tested samples of the sick leaves.
My TSWV strip came back positive! A purple or red line at the top of the strip indicates that the test worked. Another line below it, however, indicates a positive for the pathogen.
The TSWV strip shows a positive for the presence of TMV.
The TMV strip, on the other hand, testing for presence of another common tomato viral pathogen Tobacco Mosaic Virus, came back negative.
TSWV is a single-stranded RNA (ssRNA) virus in the genus Tospovirus. Viruses in this genus are transmitted to crops by an insect vector, most commonly thrips, which are slender and winged and prefer dry weather. In spite of its name, TSWV has been known to infect other crops, including peanut, tobacco, and pepper. Its destruction and economic impact can be significant in the American Southeast, where susceptible crops are commonly grown and harvested. Stunted growth, necrosis, and chlorosis are common symptoms, as well as curling and distortion of leaves, as was observed in the garden.
Thursday, June 26th
Thursday’s lecture was dedicated to one of my favorite topics in biology: viruses. A virus is an obligate parasite, as it can only replicate inside of a host cell. Up to 7,400 different viruses can infect plants, and they cannot be eradicated. The virus finds its host via a vector, attaches to a cell, and injects it with its genetic material- DNA or RNA, in the case of retroviruses. The genetic material enters the nucleus of the plant cell and uses the cell’s transcription machinery (such as reverse transcriptase, which must convert RNA from a retrovirus to a DNA template.) The cell can then translate the viral proteins, such as the capsid and envelope, and these pieces assemble with the copied genetic information to form new viral particles.
Symptoms of a virus include stunted growth, chlorosis (yellowing), or necrosis or lesions. Some viruses can spread to and block the plant vasculature and prevent transport of water or nutrients to tissues. This was observed in the gardens, as the tomato that tested positive for the TSW virus displayed wilting and stunted growth of its foliage.
A commonly studied virus is the Tobacco Mosaic Virus. TMV requires only three genes to function: one for replicase, one for a movement protein, and another that encodes a capsid. With these three genes alone, the virus can successfully infect and replicate inside a single cell, spread to adjacent cells, and then make its way throughout the entire organism by vasculature or to other plants through the stomata. Due to the efficiency and high replication rate of plant viruses, they serve as promising vectors for gene therapy.
Another Alternaria observation ended the day’s activities.
Friday, June 27th
A busy day in the lab began with measurements of the lesions left by the Alternaria infections. For each genotype, the color (brown or beige) and size (in millimeters) of each lesion were recorded. The lesions could be categorized by size range: less than 2 mm, between 2 and 3 mm, and more than 3 mm, and by color: brown or beige. A chart comparing the lesions between the wild-type col-O and mutant pad2 genotypes could have been produced to draw conclusions about the immunity of each strain.
Next, we prepared to plate infected samples from the garden to isolate potential bacterial and/or fungal pathogens.
For the bacterial isolation, four squares of infected tissue were cut, sterilized slightly with Clorox, rinsed in water, and added to a small tube containing water and a grinding bead to be ground to a homogenous, green, plant/pathogen solution.
One milliliter of the solution was added to a conical tube and diluted twice, resulting in 1:10, 1:100, and 1:1000 solutions of pathogen.
Three YMD agar plates were collected and designated for each of the three dilutions. One-half of a milliliter of each solution was placed on its designated plate, and, with the help of glass beads, spread evenly across the agar.
The next step was to plate infected tissue from the same sample on V8 plates, with the goal of isolating fungal spores, if present in the plant. Just as before, four squares of tissue were cut and sterilized in Clorox, then placed along the outside of one V8 plate. the plates were left for incubation. We hope to see our newly isolated pathogens grow next week when Outpace Week 5 returns!
This week has been one of the most exciting yet, complete with interesting lectures, hands-on lab experiences, and of course, a trip to the gardens to watch plants fight invaders!
We ended last week by inoculating our Arabidopsis thaliana plants with the hemibiotrophic bacterial pathogen Pseudomonas syringae. We used two genotypes of Arabidopsis—the wild type, Columbia-0, and a mutant lacking an important gene required for pathogen resistance, npr1. After the weekend, we spent a few days analyzing the results of the infection.
Visually, the two genotypes showed a marked difference in the level of infection. As expected, the resistant wild type plants looked healthier than the devastated susceptible plants, which had several wilted, dead leaves. We learned several lab techniques that allowed us to quantify these results into mathematical, scientifically usable data.
First we took random, equivalently-sized samples from the leaves of both plant genotypes using a regular hole puncher, suspended them in solution (MgCl2), and ground the leaf discs until the mixture was homogenized. Then we diluted the bacterial concentration and pipetted the solution onto KB plates.
A few days later we were able to count individual colonies of bacteria to compare the amount of bacteria that infiltrated the leaves and thus how sick each genotype became. We were able to graph these results for an easy visual comparison (Fig. 1).
Fig. 1 Graph comparing the bacterial colonies observed on Columbia-0 and npr1.
This marked the end of our first full-fledged experiment. We used our observations in the UAB Community Gardens to identify possible pathogens by recognizing symptoms of disease (yellow or brown tissue, spots, stunted growth, etc). Next we took samples of infected tissue (admittedly, this part we did in the lab with lab-grade plants after we purposefully infected them) and analyzed the pathogenicity of the bacteria. An important part of a plant pathologist’s job is to determine how different genes are involved in resistance and immune response. We did this by using two genotypes, Columbia-0 and npr1, and were able to see that the NPR1 gene is involved in resistance—without it, the plants are much more susceptible to disease.
However, OUTPACE isn’t all work and no play. Following the conclusion to our experiment, we played a plant-versus-pathogen board game called Vegevaders. This 2-player game is designed to let users experience the co-evolution between plants and pathogens and understand the basic pathways of pathogenesis and plant immune response. More information, as well as materials for the game, can be found here.
To make our game more competitive, candy prizes were handed out at the end of the session.
To bring this week full-circle, we left off by doing another round of infections, except we used a necrotrophic fungal pathogen called Alternaria brassicicola. Unlike our bacterial infections, we did not inoculate the pathogen through the stomatal openings on the undersides of the leaves. Instead we collected spores from fungal cultures grown on V8 plates,
These are the fungal culture plates used to obtain spores for the infection.The top and left plates have already been harvested. The bottom plate is in the process of collecting, and the right plate is waiting to be collected.
Then we counted the spores to make sure the concentration was high enough for an infection,
and pipetted 20μl of spore solution (spores suspended in potato dextrose) directly on top of the leaves. The fungus will be able to infiltrate the leaves on its own by piercing through the cuticle and underlying tissue. We used Columbia-0 as our wild type again, but we used pad2-1 as our susceptible mutant. Next week we will be able to analyze the results from this experiment and compare them to the bacterial infection. It will be interesting to see the differences in how these two pathogens affect the plants and how the two strategies—biotrophic and necrotrophic—vary in terms of pathogenicity and speed of infection.
We collected similar-sized leaves from the plants (Columbia-0 on the left, pad2-1 on the right) and transferred them to a plate for infection.
Close-up on the plate before the leaves are infected.
Here is a post from the Plant Pathogen Detective, Brenna Terry:
Symptoms on the infected cucumber plant
The healthy neighbor
Brenna is right about her diagnosis.
Plants infected with Alternaria cucumerina usually develop circular spots or lesions on the oldest or crown leaves near the center of the hill. The number of spots increases rapidly in warm, humid weather, later spreading to the younger leaves toward the tips of the vines. At first the lesions are small, circular, and somewhat water-soaked or transparent. They enlarge rapidly until they are 1/2 inch or more in diameter, turning light brown when mature. Definite concentric rings may often be seen in the older leaves, round to irregular spots, giving them a target like appearance. Spots may merge, blighting large areas of the leaf (as seen in Brenna’s second picture). The leaves commonly curl, wither, and fall prematurely.
We’ll perform Alternaria infections at the end of this week and then we’ll learn more about this devastating fungus! Feel free to share stories and photos of your sick plants!
A slide from Dr. Mukhtar’s lecture on P. syringe infection protocol.
We’re starting the infection by spinning down the bacterial culture and diluting the pellet to a required Optical Density (OD).
Each of us got 36 plants to infect. Half of them were wild type resistant plants Columbia-0 and the other half were the hypersusceptible npr1 mutant plants. We marked leaves to be infected using permanent markers.
Then we pressure-infiltrated the marked leaves using small needleless syringes. The inoculated plants went back to the growth room and we will check them out in three days to evaluate symptoms and take samples for colony count.
It was a fun experiment!
I just received a very thoughtful email from one of our fellow students, Maggie McCormack. Please feel free to post your comments to her questions and we will discuss this very important aspect tomorrow!
"Hi Dr. Mukhtar,
I’ve been thinking about yesterday’s lecture and I had a few questions.
I remember from your plant biology class and from working in the lab that seeds require a cold period to induce germination. In lecture you mentioned that many pathogenic bacteria do not survive cold temperatures. Could the plant’s need for cold weather before germination be a kind of preemptive defense mechanism (ie. if they wait for a period of cold, it ensures that the soil and surrounding area is sterilized and they have a better chance of growing before being attacked by bacteria)?
I’m also curious about the systemic required resistance. Does this signal occur only within the host plant or can a plant signal nearby plants as well? It seems especially useful for plants such as grasses that propagate through runners. If a plant like that became infected, wouldn’t it put all the plants connected to it at risk as well?
I’m really enjoying this experience—thank you so much for letting me be a part of it!”
Mildew on a cucumber plant
This is a great picture of the insect babies or eggs getting attached to the plant. Focus on the red dots.
This is a great example of a plant getting overtaken by Mildew. You can see all the white on the plant.
We were busy today preparing media and pouring plates for cultivation of various phytopathogenic microbes.
Alison and Darion prepared V8 juice plates that are used to promote sporulation of various fungi.
One of the key ingredients of this medium is the well known Campbell’s V8 vegetable juice!
Brenna and Audrey prepared YDC media that is used for subculturing suspected bacterial pathogens xanthomonads and clavibacters.
Maggie and Cody made YPD media that is a rich source of food for many plant pathogenic bacteria.
We will use our newly prepared media to culture phytopathogenic microbes identified in the UAB Gardens in the coming weeks!
Today we made our first trip to the Gardens!
We took a group photo…
…and found several interesting examples of different infections!
We identified typical symptoms of a viral infection…
…spotted a bean plant with symptoms of the bacterial leaf spot disease (caused by Xanthomonas campestris pv. vesicatoria)…
…and another bean plant with symptoms of infection by the Bean Mosaic Virus:(
Here is a zucchini plant suffering from a powdery mildew infection. It is typically caused by the fungal species Podosphaera xanthii, but may also be caused by the fungi Golovinomyces cucurbitacearum and Golovinomyces orontii.
This pepper plant is infected with another fungal pathogen Septoria lycopersici (Septoria blight disease).
We’ll be back in the Gardens next week to ID some of these pathogens with immunodetection kits!