10.5061/DRYAD.6DJH9W10M
Getman-Pickering, Zoe
0000-0002-5695-858X
George Washington University
Stack, George
Cornell University
Thaler, Jennifer
Cornell University
Fertilizer quantity and type alter mycorrhizae-conferred growth and
resistance to herbivores
Dryad
dataset
2021
FOS: Agricultural sciences
United States Department of Agriculture
https://ror.org/01na82s61
1008468
National Science Foundation
https://ror.org/021nxhr62
DGE-1650441
The Cornell Mellon Fund*
Cornell Entomology Griswold Fund*
The Cornell Mellon Fund
Cornell Entomology Griswold Fund
2021-01-04T00:00:00Z
2021-01-04T00:00:00Z
en
46805 bytes
3
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.
We conducted a 2x5 factorial greenhouse experiment to test the interacting
effects of mycorrhizae and five fertilizer treatments on tomato plant
resistance to herbivores. Study system We conducted these experiments
using tomato plants var. Castlemart (Solanum lycopersicum). 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 (Trichoplusia ni) 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® 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. Experimental conditions 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. Measurements
Mycorrhizal Colonization 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. Plant Growth 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. Resistance to herbivores
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 Trichoplusia ni
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 (mm2) using a 4mm2 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. Resistance Traits 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 T. ni (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 (>100%) were removed.