Experimental design
We ran a 2x2x2 fully factorial randomized design with plants grown
singly or in competition, with or without mycorrhizae, and with or
without induction of jasmonate defenses (n=~24 per
treatment, n=191 total).
Castlemart tomato seeds were surface sterilized using a solution of 30%
of household bleach in distilled water for 30 minutes. They were then
rinsed for 1 minute under running water. We sterilized 2.5x3 cm peat
pots, 10 cm plastic pots, and 1:1 sand and turface media (by volume) in
an autoclave at 121℃ at 1 bar of pressure for 90 minutes. This was
repeated 3 times with a minimum of 24 hours between each autoclaving.
Due to uncertain germination rates, we planted 2-4 tomato seeds into
each peat pots filled with either sand-turface mixture and mycorrhizal
inoculum or control inoculum in a 3:1 ratio. Following
germination, we established our competition and no competition
treatments by thinning all treatments so that each pot had either one or
two seedlings.
After the tomato plants had germinated and had been thinned, we
transferred the peat pots into 10 cm pots that were filled with a 1:1
mixture of sand and turface. Each individual pot was placed in a petri
dish to prevent mycorrhizal contamination between plants. We randomized
the position of the plants on the greenhouse bench. Plants were watered
as needed using tap water and fertilized with 21-5-20 NPK fertilizer
diluted to 6ppm every 10-15 days. When the tomato plants were 50 days
old, half of them were sprayed with approximately 0.3 g of a 0.5 mM
jasmonic acid solution dissolved in a 4% ethanol solution to simulate
insect herbivory and induce defenses. Control plants were sprayed with
0.3 g of a 4% ethanol solution. Three days after the application of
jasmonic acid, we measured plant height as a metric of plant vigor and
harvested leaf tissue for bio and chemical assays. Biomass was not
collected as most plant tissue was harvested for assays.
To determine the effect of competition and mycorrhizae on resistance to
herbivores, we harvested the terminal leaflet from the second fully
expanded leaf using a clean razor blade and placed it in petri dishes on
moist filter paper for a bioassay. First-instar cabbage looper
caterpillars (Trichoplusia ni ) were placed on the leaves and
allowed to feed. After 3 days, caterpillars were weighed to determine
changes in herbivore mass in each of the eight treatments (Thaler and
Bostock 2004). We also measured plant damage by measuring leaf area
consumed using the grid method (Coley 1983).
To test the potential mechanisms of altered resistance we measured the
constitutive and induced resistance traits: Protease inhibitors and C/N
ratio. Protease inhibitors are a class of chemical defenses that reduce
the digestibility of leaf tissue by breaking down the herbivore’s
digestive enzymes. Protease inhibitors are produced through the jasmonic
acid pathway and can be used to measure expression of this pathway. The
C/N ratio on the other hand provides information on both the health of a
plant, with a low ratio correlated to healthier, plants as well as its
attractiveness to herbivores. Most herbivores are nitrogen limited
(White 1984), so plant tissue with a high C/N ratio can be less
attractive and nutritious (Behmer 2009). Additionally, plants can lower
nitrogen levels in tissue in response to herbivory to deter herbivores
and protect valuable resources (Newingham et al. 2007; Gómez et al.
2010).
We took the terminal leaflet from the first fully expanded leaf from
each plant for analysis using colorimetric protease inhibitor assays
(Orians et al. 2000). The remaining leaf tissue was dried and
homogenized using 2.3mm zircon beads (RPI) in a ball grinder. Then, 0.8
mg of ground leaf tissue was balled into 4x6mm tin capsules and analyzed
using an Elementar analyzer (CHNS) to determine carbon and nitrogen
levels.
The soil was dried prior to harvesting roots from each plant to measure
levels of mycorrhizal colonization. Roots were stored in ethanol until
they could be stained following the ink and vinegar method (Vierheilig
et al. 1998). Following staining, samples were stored in a 50%
glycerol, 45% water, 5% 1.65 M HCl solution until mycorrhizal
colonization was confirmed using microscopy.