DOI: 10.5061/dryad.6djh9w10m
title: Fertilizer quantity and type alter mycorrhizae-conferred growth and resistance to herbivores

description: 1. Plants face a constant struggle to acquire nutrients and defend
 themselves against herbivores. Mycorrhizae are fungal mutualists that
 provide nutrients that can increase plant growth and alter resistance to
 herbivores. The beneficial effects of mycorrhizae for nutrient acquisition
 can depend on the quantity and type of soil nutrients available, with
 plants usually benefiting more in terms of growth from mycorrhizae when
 nutrients are limited. However, it is unclear how the addition of
 different nutrients might shift mycorrhizal conferred resistance to
 herbivores by changing defensive secondary chemistry and nutrient
 availability. 2. We conducted two concurrent greenhouse experiments to
 test how three levels of fertilizer (low, medium, and high) and three
 types of fertilizer (organic, organically derived, and inorganic) altered
 mycorrhizae-conferred resistance to herbivores in tomato plants. In
 addition, we looked at whether mycorrhizae-conferred resistance was driven
 by plant secondary metabolites or the nutrient content of the leaves. 3.
 Association with mycorrhizae was associated with an increase in biomass at
 low levels of fertilization and decreased biomass at high levels of
 fertilization. Interestingly, mycorrhizae increased resistance to
 herbivores at medium levels of fertilization, but had no effect at low and
 high levels of fertilization. Mycorrhizae improved resistance most
 strongly when plants were fertilized with a high phosphorus, organically
 derived fertilizer. In both experiments, increased resistance was
 correlated with changes in the plant’s foliar nitrogen content. 4.
 Synthesis and applications: Our study supports the potential for
 mycorrhizae to improve either crop growth or pest resistance under lower
 fertilizer conditions. However, mycorrhizae did not provide both growth
 and resistance benefits under any treatment. While mycorrhizae have the
 potential to benefit crops in in lower input systems, it may be
 challenging to maximize both growth and resistance benefits.
description: We conducted a 2x5 factorial greenhouse experiment to test the
 interacting effects of mycorrhizae and five fertilizer treatments on
 tomato plant resistance to herbivores. <b>Study
 system</b> We conducted these experiments using
 tomato plants var. Castlemart (<i>Solanum lycopersicum</i>).
 Tomatoes are a valuable field and greenhouse crop that associate with
 mycorrhizal fungi and have a range of chemical defenses against
 herbivores. The defensive chemistry of tomatoes has been well
 characterized, and protease inhibitors in particular have been identified
 as important for defense (Felton, Broadway, & Duffey, 1989;
 Shrivastava et al., 2015). We used first instar cabbage
 looper (<i>Trichoplusia ni)</i> caterpillars in a bioassay to
 measure plant resistance. Cabbage loopers are generalist noctuid
 caterpillars that feed on a wide variety of crop plants including
 solanaceous and cruciferous vegetables. They were chosen because they are
 sensitive to changes in host plant quality. We used them in their first
 instar because this stage is most sensitive to plant defenses (Thaler
 unpublished data), and because later instar caterpillars may have
 developed resistance to plant defenses if they were fed on leaves (Lee
 & Berenbaum, 1989). These larvae were obtained from a colony
 maintained on artificial cabbage looper diet (Southland Products Inc.) at
 Cornell University for many years. In both experiments,
 we used mycorrhizae extracted from soil collected at the Dilmun Hill
 student organic farm at Cornell University (Ithaca, NY). Diverse, field
 collected mixtures have been found to be more beneficial to plants than
 monocultures (Rowe, Brown, & Claassen, 2007; Rúa et al., 2016),
 and are more representative of the conditions crop plants experience in
 the field. To isolate mycorrhizal spores, the soil was wet sieved to
 remove large rocks and debris and then blended for 20 seconds using a
 Cuisinart™ immersion blender. The resulting liquid was passed through a
 series of sieves with the smallest having a pore size of 600 µm. We then
 used a 20 µm nylon mesh to remove excess water. The soil slurry was
 divided into 5 mL aliquots and resuspended in 40 mL of a 30% sucrose
 solution. This mixture was centrifuged in a bucket attachment at 2200 rpm
 for 2 minutes. The supernatant was decanted through a 30 µm mesh set over
 a funnel. The spores on the mesh were washed with 20 mL of DI water into a
 beaker. Spores were filtered through 30 µm mesh,
 surface sterilized with a solution of 4% chloramine T, 0.05%
 Tween<b><sup>®</sup></b> 20, 0.02% Gentamicin and
 0.01% Streptomycin using the methods outlined by Mukerji et al., 2002
 (page 305). We used microscopy to determine that extracted spores were
 clean and contained a diverse range of morphologies. The spores were
 resuspended in DI water such that 10 µL of water contained between 10-12
 viable spores. The solution was kept suspended using a vortex during
 application. Two weeks after the plants germinated, half were inoculated
 using 100 mL of the spore solution pipetted at the base of the plant and
 watered down. The control plants were treated with 100 mL of DI water and
 also watered down. To recover soil microbes, which could be have an impact
 on both plant health (Berendsen et al. 2012 and references there in),
 mycorrhizal fungi (Desirò et al 2014), and the interaction between the two
 (Artursson, Finlay, & Jansson, 2006), we filtered a mixture of
 Lambert LM-AP potting soil and water through a 1 µm sieve, and added 20 mL
 of the resulting solution to each pot. We used potting soil to reduce the
 risk of introducing pathogenic species.
 <b>Experimental conditions </b> We
 grew 220 Castlemart tomato plants in individual 10 cm pots filled with a
 1:1 sand and calcined clay media. The media was autoclaved for one hour 3
 times, 24 hours apart at 121°C to sterilize it before use. The tomato
 seeds were surface sterilized for 15 minutes in a 15% household bleach
 solution and then rinsed under running water for 1 minute. The plants were
 grown at 34 °C and watered with 60 mL of water every 2-4 days.
 We divided the plants into two concurrent experiments. In the
 first, we tested the effects of mycorrhizae and different amounts of
 inorganic fertilizer. Plants were treated with a low, medium, or high dose
 of inorganic 21-5-20 NPK fertilizer (Table 1). We chose this low
 phosphorus fertilizer to encourage association with mycorrhizal fungi.
 Guaranteed Analysis for all fertilizers is available in Supporting
 Information 1. Sixty plants were given a low dose (20 mL) of a 21-5-20
 fertilizer diluted to 150 mg/L. Another 60 plants were given a medium dose
 (30 mL) of the same fertilizer. A third set of 60 plants were given a high
 dose (40 mL) of the same fertilizer. Each plant was given supplemental
 water such that each plant received an equal amount of water.
 To test the effect of fertilizer type, we compared three types of
 fertilizer: an organic (n=20), organically derived (n=20), and inorganic
 commercially available fertilizer (n=60). Guaranteed Analysis for all
 fertilizers is available in Supporting Information 1. We chose three
 commonly used fertilizers and applied them as recommended on the label. We
 chose not to adjust quantity of fertilizer to equalize the N or P level
 because differences in macronutrient accessibility and micronutrient
 levels would limit our ability to link effects to a single
 macronutrient. In the organically derived treatment, 20
 plants were fertilized using a higher phosphorus fertilizer: Foxfarm Grow
 Big liquid plant food 6:4:4 diluted to 4 mL/L of fertilizer. In the
 organic treatment, 20 plants were fertilized with the organic, carbon-rich
 Alaska brand fish fertilizer 5:1:1 diluted to 14.3 mL/L water as
 recommended. For the inorganic treatment, we used the same plants that
 were given a high dose (40 mL) of the 21-5-20 fertilizer from experiment
 1. We fertilized the plants grown in the 21-5-20 fertilizer once every two
 weeks, while the other two fertilizers were applied once every 4 weeks, to
 maintain a more comparable total nutrient addition.
 <b>Measurements</b>
 <i>Mycorrhizal Colonization</i> To
 confirm mycorrhizal colonization, we bleached the roots using potassium
 hydroxide and stained the roots using Schiffer black ink (Vierheilig et
 al. 1998). Using microscopy, we assessed the roots to confirm that plants
 in the mycorrhizal treatment were colonized and those in the control were
 not. Previous work by Rutkowski et al. (unpublished) found that variation
 in colonization had no effect on resistance to herbivores or resistance
 traits. We had no accidental colonization in the control
 treatment. <i>Plant Growth</i>
 We harvested the plants two months after germination. We excised
 the terminal leaflet from the most recently fully extended leaf for
 protease inhibitor analysis and from the second most recently fully
 extended leaf for the bioassay. We harvested and dried the remaining leaf
 and stem tissue for 1 week to measure dry biomass and to analyze for
 carbon:nitrogen ratio (C:N ratio), a measure of the nutritional content of
 plants. <i>Resistance to herbivores
 </i> To measure herbivore performance, we excised
 the terminal leaflet from the third most recent fully extended leaf and
 placed it in a 9 cm petri dish lined with damp filter paper. We placed 2
 neonate <i>Trichoplusia ni</i> caterpillars on each leaflet,
 closed the petri dish, and sealed the petri dish with parafilm. After 6
 days, we measured the mass of each caterpillar. We observed that many
 caterpillars left the leaf and died. We recorded the number of such
 caterpillars as a metric of repellence. Caterpillars that died without
 moving or feeding were removed from analyses as we presumed that they were
 killed during transportation to the leaf. We also measured levels of
 herbivory (mm<sup>2</sup>) using a 4mm<sup>2</sup>
 grid to assess the quantity of leaf consumed (Coley 1982).
 Using excised leaves to measure herbivory and resistance is a
 common approach (Kumar, Ortiz, Garrido, Poveda, & Jander, 2016; J
 S Thaler, Stout, Karban, & Duffey, 1996; Jennifer S. Thaler,
 Agrawal, & Halitschke, 2010) that prevents confounding temporal
 variation (Karban, 2011). However, removing the leaves may differentially
 induce resistance to herbivores in the mycorrhizal and non-mycorrhizal
 plants (Pozo and Azcón-Aguilar, 2007 and citations therein). Induced
 defenses are resistance traits that are deployed only once the plant has
 been damaged, while constitutive defenses exist regardless of damage. We
 are unable to differentiate whether the effects of mycorrhizae on
 resistance were due to changes in constitutive or induced resistance. If
 mycorrhizae are affecting induced, but not constitutive resistance, it may
 mean that herbivores will be able to damage plants in the field
 significantly before the defenses are deployed.
 <i>Resistance Traits</i> We
 measured plant nutritive quality using C:N ratio. The C:N ratio is
 indicative of both the attractiveness of a plant to herbivores and the
 health of a plant, with a low ratio correlated to healthier, more
 fertilized plants. Most herbivores are N limited (White, 1984), so plants
 with a low C:N ratio can be more attractive and nutritious (Behmer, 2009).
 To test the role of mycorrhizae and fertilizer on leaf nutrient quality
 and the effect of leaf nutrient quality on herbivory, we analyzed the C:N
 ratio of one leaf per plant. Each leaf was ground into a powder using
 2.3mm an Mp Biomedical Fastprep 24. Then 5 ± 0.1 mg tissue from each leaf
 was balled into 4x6 mm tin capsules (Costech Analytical Technologies Inc)
 and analyzed using a Costech 4010 CHNS-O Analytical Combustion
 System. We measured plant chemical defense by measuring
 protease inhibitor activity. Protease inhibitors are a class of chemical
 defenses that reduce the digestibility of leaf tissue by breaking down the
 herbivore’s digestive enzymes (Chen, Wilkerson, Kuchar, Phinney, &
 Howe, 2005). In tomatoes, they play a strong role in the resistance to
 herbivores including <i>T. ni </i>(Scott, Thaler, &
 Scott, 2010). Protease inhibitors are produced through the jasmonic acid
 pathway and can be used to measure expression of this pathway (Koiwa,
 Bressan, & Hasegawa, 1997). Mycorrhizae have been shown to alter
 protease inhibitor levels under different conditions (Barazani 2004). We
 excised the terminal leaflet from the most recent fully extended leaf and
 immediately froze it on dry ice. We analyzed 100mg of tissue using a
 colorimetric assay to calculate the activity of defensive trypsin protease
 inhibitors using a method adapted from Hegedus et al. (2003) (Supporting
 Information 2). Data points with erroneous values (&gt;100%) were
 removed.