how would the increased moisture impact the environment during shipment?
(a) The availability of water is a critical environmental variable for disease development. Consider creating different
humid environments with damp paper towels, plastic
bags, or airtight containers.
(3) Alter light exposure. After buying potatoes at the supermarket, they are often stored in a dark pantry. What may happen
if the potatoes are stored in sunlight or under classroom lights
compared to the dark?
(a) Although less well characterized by scientists, consider
altering the exposure to light (dark vs. florescent vs.
sunlight) or the color of the light by wrapping containers in different colors of plastic.
(1) Alternate sources of inoculum. Bacterial soft rot pathogens
are often adapted to their hosts. Given soft rot bacteria from
different hosts, which do you think will most likely be able
to infect potatoes and why?
(a) Gather inocula from different hosts, and inoculate on
disinfested potatoes. For example, collect inocula from
bell pepper, carrot, and cucumber utilizing the same technique as outlined for the core protocol and investigate
which pathogen is most virulent on potato. Inocula from
non-potato hosts can be inoculated into healthy potatoes
utilizing the protocol outlined previously. Enterobacteriaceae are known to infect numerous varieties of plants,
and there are many resources that list the known host
species (Bhat et al., 2010).
The most practical consideration with this laboratory exercise is
managing the odor during incubation. Since disease symptoms are
best analyzed between five and seven days after inoculation, culture
incubation in a well-ventilated area is encouraged. For example, consider incubating potatoes in a chemical fume hood (if available) or
near an open window with an exhaust fan. Furthermore, the odor
in the classroom should be mitigated prior to class because disruptive behavior (especially for middle school students) often increases
in laboratories with pungent odors. Although the bacteria cultured
in this experiment are not human pathogens, instructors should
exhibit best laboratory practices, including providing eye protection,
gloves, and protective garments for the students. Since soft rot bacteria are naturally occurring, most locations do not require special
Expect to observe a variety of results during this laboratory experiment, since each lab group will inoculate and incubate their potatoes
differently. For example, humidity is a critical variable for disease
development, and some lab groups may not adequately wet their
paper towels or sufficiently seal their plastic bags prior to incubation.
Lack of sufficient moisture often results in asymptomatic potatoes,
even when inoculated with the pathogen. Also, symptoms exist on a
spectrum and often appear differently between inoculations. Common symptoms are tissue softness (superficial or very deep within
the potato) and discoloration (tan to nearly black; Figure 2B, C).
These symptoms should not be confused with hollow heart, which
is a blackening of the center of the potato in response to increased
moisture, or cork formation, which is a natural scab-like tissue layer
that often surrounds the toothpick wound when viewed on a potato
cross section (Figure 2C, D). Lastly, fungi often contaminate wounded
potatoes and are visible as a fuzzy mass commonly associated with the
inoculation site (Figure 2A). These fungi are most likely not causing
disease, but rather feeding on the dead potato tissue.
The microscopic analysis of infected tissue often yields unclear
results due to insufficient magnification. Prior to viewing a diseased
sample, consider instructing the students to view healthy potato tissue first so they become familiar with the components of potato
cells. The most visible component of potato cells without specialized
staining are the amyloplasts, which are the plastids that store starch
(Figure 3). Each potato cell contains numerous amyloplasts, which
appear as bean-shaped structures with dark margins. In the healthy
sample, the liquid between the amyloplasts should be clear. When
viewing the diseased sample, remember that bacteria are small (
generally 1–2 µm in length) and are often difficult to observe individually. Bacteria are only faintly visible at 400× magnification and may
require dimming the illumination on the microscope to identify
them. Since the infected tissue should contain vast quantities of bacteria, the pathogens should be visible as an opaque, shifting layer
(resembling television static) between amyloplasts.
Students often experience difficulties with the nuanced vocabulary associated with plant pathology, but an explanation of the following words and their definitions will likely lead to increased content
Pathogens vs. disease. Diseases are disruptions in normal cellular
function. Diseases can be caused by abiotic (nonliving) factors like
nutrient deficiencies or by biotic (living) factors like bacterial pathogens. Pathogens are the causal agents of disease. In this laboratory
protocol, the Enterobacteriaceae are the pathogens, and soft rot is
Signs vs. symptoms. Symptoms are the physiological host responses
associated with pathogen infection. For example, symptoms of infection by soft-rot-causing bacteria on potato are soft rot, discoloration,
and a pungent odor. Signs of an infection are the pathogens, which
in this experiment are bacteria that can be observed only under high
magnification on a compound microscope or as ooze flowing out of
Infect vs. infest. Infection is the initiation of a parasitic relationship with a host, whereas infestation is to reside on the exterior of a
surface without infecting. Nearly all potatoes are infested with
Enterobacteriaceae from the soil; however, potatoes can remain
healthy in storage for many weeks until the environment becomes
conducive for pathogen infection.
Pathogenicity vs. virulence. Pathogenicity is a categorical variable
( i.e., yes or no) describing whether an organism can cause disease.
Virulence is a continuous variable ( i.e., from 1 to 10) that describes
the symptom severity. For example, a species of bacteria may be
pathogenic on potatoes but exhibit moderate virulence.
The inquiry-based protocol may take numerous directions
depending on which variables are tested. When designing each experiment, ensure that mock-inoculated disinfested potatoes serve as controls for each different approach. When investigating pathogen
virulence, note that this protocol does not necessarily involve a pure
culture. When the inoculum is initially generated for the experiment,