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.