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 genusTospovirus. 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!