Ten gallons of soil were collected from each of four sites in south
Florida, on the criteria of proximity and soil type: the Redlands agricultural area, the Canal Point sugarcane area, a lakeshore near the
school, and a field near the school (Figure 1). To contrast the effects
of soil bacteria on plant traits, half of the 10 gallons of soil from each
site was sterilized by autoclaving, killing all soil microbes. Soil collection and preparation was completed by laboratory assistants and volunteers from a nearby university (Florida International University in
Miami). The project could be adapted so that students collect soil
on their own from backyards, farms, or natural areas; the goal is to
capture soils different in soil bacteria, and choosing different soils
from different habitats is the most likely way to achieve this. By comparing plant growth and rhizobial association on soils from different
sites, one can remove the need to autoclave soil if no autoclave is available. However, when available, we feel this is an excellent opportunity
to teach aseptic technique. Alternatively, simple but effective techniques have been developed using a household microwave (Ferriss,
1984; Eichenberger, 1991; Nelson and Trabelsi, 2016).
In the classroom discussions, students identified several common legumes and then chose five leguminous species to be planted
in each of the four soil types. We ended up selecting legumes that
perform well in our subtropical conditions and are popular food
crops with our large Hispanic community. However, in more temperate climates, plants such as garden peas, soybeans, or peanuts
may be more suitable (see Supplemental Material S1). Students potted and planted each plant species in each soil type in 160 mL
cone-shaped containers (Cone-tainers; SC10-R, Stuewe & Sons,
Tangent, Oregon). Cone-tainers are not essential; pots or other
containers could be used instead.
Pre-sterilized Cone-tainers were filled with soil and gently com-
pacted to within 1 inch of the Cone-tainer top. Two seeds of the
same type were planted less than 1 inch from the soil surface and
gently covered with soil. Students should be instructed to not plant
seeds too deep in the soil column, which would reduce germination
greatly. This is particularly true with small-seeded species, such as
weeds like Medicago lupulina or clovers like Trifolium repens.
Seeds of each plant species were planted with five replicates in
each type of soil: five legume species × five replicates × eight soil
types (half sterilized and the other half unsterilized) = 200 Cone-
tainers (Figure 2). Cone-tainers were arranged within Cone-tainer
racks (RL98, Stuewe & Sons). The Cone-tainers were arranged in
several large plastic tubs with a few inches of water. Each tub con-
tained Cone-tainers with a single soil type (and sterility status) to
avoid cross-contamination of soils from different areas or different
sterility. Each tub contained all plant species: five Cone-tainers of
each legume species. Thus, each plastic tub contained 25 Cone-
tainers, which in total contained all plant species, randomly
assorted in the tub, with the same soil type (Figure 2).
Students watered plants daily until seedlings developed, after which
plants were watered weekly. The plastic tubs containing the plants were
placed in a semi-shaded area, in ambient temperature and light.
Students determined the effects of soil type and bacterial sym-
bionts on plant development and functional traits by measuring
stem height, number of leaves, flower or pod formation, and chlo-
rophyll content (SPAD502 chlorophyll meter, Konica Minolta,
Japan), every two weeks for the duration of the experiment (see
Supplemental Material S3). We allowed the plants to grow for five
months, although shorter durations of just a few weeks will work.
Fully developed nodules can usually be found on legumes by two
to three weeks of age.
Root length, stem length, and fresh weight of the plant were
also recorded on fully mature plants at harvest (see Supplemental
Material S4). The time to maturation varied among species and soil
types, but plants generally matured about 16 weeks after planting.
This works well with the moderate winters of south Florida and the
large legumes like pigeon pea. However, nodules typically develop
in two to three weeks, and some legumes with short life spans,
such as black medic (Medicago lupulina, a common temperate for-
age crop and weed), can mature in five to six weeks.
Root nodules were counted, then removed for storage on a drying agent (Drierite) and refrigerated at 4°C. This allows downstream
analysis of the nodules, for teachers who want to train students to
Figure 1. Soil collection sites in southwest Florida: (A)
Homestead farm soil, (B) field soil near Pine Crest School, (C)
lake soil near Pine Crest School, and (D) Canal Point USDA soil.
Figure 2. Image of students and experimental design.