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Conditioning the soil microbiome through plant‐soil feedbacks suppresses an aboveground insect pest

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•Soils and their microbiomes are now recognized as key components of plant health, but how to steer those microbiomes to obtain their beneficial functions is still unknown. Here, we assess whether plant‐soil feedbacks can be applied in a crop system to shape soil microbiomes that suppress herbivorous insects in aboveground tissues. •We used four grass and four forb species to condition living soil. Then we inoculated those soil microbiomes into sterilized soil and grew chrysanthemum as a focal plant. We evaluated the soil microbiome in the inocula and after chrysanthemum growth, as well as plant and herbivore parameters. •We show that inocula and inoculated soil in which a focal plant had grown harbor remarkably different microbiomes, with the focal plant exerting a strong negative effect on fungi, especially arbuscular mycorrhizal fungi. Soil inoculation consistently induced resistance against the thrips Frankliniella occidentalis, but not against the mite Tetranychus urticae, when compared with sterilized soil. Additionally, plant species shaped distinct microbiomes that had different effects on thrips, chlorogenic acid levels in leaves and plant growth. •This study provides a proof‐of‐concept that the plant‐soil feedback concept can be applied to steer soil microbiomes with the goal of inducing resistance aboveground against herbivorous insects.
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Conditioning the soil microbiome through plantsoil feedbacks
suppresses an aboveground insect pest
Ana Pineda
1
, Ian Kaplan
1,2
, S. Emilia Hannula
1
, Wadih Ghanem
1,2
and T. Martijn Bezemer
1,3
1
Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen 6700 AB, the Netherlands;
2
Department of Entomology, Purdue University, West
Lafayette, IN 47907, USA;
3
Institute of Biology, Section Plant Ecology and Phytochemistry, Leiden University, Leiden 2300 RA, the Netherlands
Author for correspondence:
Ana Pineda
Tel: +31 317 473 607
Email: a.pineda@nioo.knaw.nl
Received: 9 November 2019
Accepted: 4 December 2019
New Phytologist (2020) 226: 595–608
doi: 10.1111/nph.16385
Key words: below-aboveground, herbivores,
microbeplantinsect interactions,
microbiome-induced systemic resistance,
plantsoilinsect feedbacks, soil
microbiomes, sustainability, thrips.
Summary
Soils and their microbiomes are now recognized as key components of plant health, but
how to steer those microbiomes to obtain their beneficial functions is still unknown. Here, we
assess whether plantsoil feedbacks can be applied in a crop system to shape soil microbiomes
that suppress herbivorous insects in above-ground tissues.
We used four grass and four forb species to condition living soil. Then we inoculated those
soil microbiomes into sterilized soil and grew chrysanthemum as a focal plant. We evaluated
the soil microbiome in the inocula and after chrysanthemum growth, as well as plant and her-
bivore parameters.
We show that inocula and inoculated soil in which a focal plant had grown harbor remark-
ably different microbiomes, with the focal plant exerting a strong negative effect on fungi,
especially arbuscular mycorrhizal fungi. Soil inoculation consistently induced resistance against
the thrips Frankliniella occidentalis, but not against the mite Tetranychus urticae, when com-
pared with sterilized soil. Additionally, plant species shaped distinct microbiomes that had dif-
ferent effects on thrips, chlorogenic acid concentrations in leaves and plant growth.
This study provides a proof-of-concept that the plantsoil feedback concept can be applied
to steer soil microbiomes with the goal of inducing resistance above ground against herbivo-
rous insects.
Introduction
Soils are crucial for terrestrial life (Wall et al., 2015). The soil is
where most terrestrial plants start their growth and, more than a
simple substrate, is home to a diverse community of microbes.
Several of these microbes provide plants with key functions, such
as enhanced growth via improved nutrition or suppression of soil
pathogens (Pieterse et al., 2016; Raaijmakers & Mazzola, 2016).
An important soil service is the protection of above-ground plant
tissues against pests and diseases (Bardgett & Wardle, 2010),
and this could be used to improve sustainability in agriculture
(Kaplan et al., 2018; Mariotte et al., 2018). The soil is the source
of most beneficial microbes that colonize the rhizosphere (Bul-
garelli et al., 2013) the thin interface of root surface with
attached soil which are key players in plant immunity and
overall plant performance. To date, soil management typically
focuses on avoiding detrimental effects such as allelopathy or
accumulation of pathogens and pests via crop rotation (Peralta
et al., 2018). However, an exciting possibility is to manage soils
to steer microbial communities to a desired beneficial state with
a focus on promoting the presence and activity of beneficial
microbes, instead of simply avoiding the pathogenic ones.
Empirical evidence that this type of soil management can
increase resistance in crops against above-ground herbivores is,
however, still lacking (Pineda et al., 2017).
It is well established that rhizosphere colonization by beneficial
soil microbes can reduce the negative impact of above-ground
herbivores on plant growth (Pieterse et al., 2014; Pineda et al.,
2017; Rashid & Chung, 2017). For example, soil microbes can
prime plants to respond faster or stronger to their attackers, espe-
cially to cell-feeding and leaf-chewing herbivores (Martınez-Med-
ina et al., 2016). Until recently, research on microbeplant
insect interactions has focused on the effects of a limited number
of individual microbial strains, which often generate inconsistent
results when applied in the field (Gadhave et al., 2016; Timmusk
et al., 2017). An alternative approach is to focus on the complete
microbiome. Several authors have argued that the introduction of
more complex soil communities, rather than single species/
strains, is necessary to achieve consistent enhancement of crop
protection (Busby et al., 2017; Pineda et al., 2017), but so far,
evidence of resistance against herbivores triggered either by such
microbiome or by a single microbial strain functioning in a com-
plex microbial community is scarce. Interestingly, studies with
Arabidopsis or other wild plant species have shown, using soil
sieving or sterilization, that the soil microbiome as a whole plays
a significant role in inducing plant resistance to leaf-feeding her-
bivorous insects (Badri et al., 2013; Hubbard et al., 2019; Wang
et al., 2019). Until now, most studies on soil microbiomes have
focused on building synthetic communities based on culturable
organisms (Santhanam et al., 2015; Herrera Paredes et al., 2018)
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Research
and the challenge is now how to manipulate those microbiomes
to optimize induced resistance in crops.
One approach to steer soil microbiomes is to apply the ecologi-
cal concept of plantsoil feedbacks (van der Putten et al., 2013;
Bennett & Klironomos, 2018). Plants release primary and sec-
ondary metabolites through their roots that shape the soil and
rhizosphere microbiome in a species-specific way (Hu et al.,
2018; Yuan et al., 2018). Thus, when a new plant grows in soil in
which another plant had previously grown, its performance can
be enhanced or reduced, depending on changes in the soil trig-
gered by the first plant. Plantsoil feedbacks are well studied in
the context of succession and invasion ecology to explain how dif-
ferent plant species interact (van der Putten et al., 2013).
Notably, several recent studies indicate that plantsoil feedbacks
also affect above-ground plantinsect interactions in wild plant
species (Kos et al., 2015; Heinen et al., 2018; Hannula et al.,
2019). An important question is whether and how plantsoil
feedbacks can be used to steer soil microbiomes to increase resis-
tance of later-growing crops to above-ground pests (Kaplan et al.,
2018; Mariotte et al., 2018).
Soil inoculation has been mainly studied in the context of
restoration ecology, showing that degraded soils and their
ecosystem functions can be restored quicker when soil micro-
biomes are inoculated (Middleton & Bever, 2012; Wubs
et al., 2016). Agricultural fields and especially glasshouse soils
are highly degraded, but the application of soil inoculation to
restore the above-ground functions of these soils is a new
research field. Recently, we showed how inoculation with soils
in which grasses had previously grown generally increased
growth of chrysanthemum in the presence of the soil
pathogen Pythium (Ma et al., 2017). This study supports the
idea of applying the concept of plantsoil feedbacks to
enhance the soil suppressiveness against pathogens (Schlatter
et al., 2017). An important question that now needs to be
answered is which part of the soil microbiome is altered by
the first plant, and the extent to which these changes relate
to the growth of the second plant. Plantsoil feedbacks are
not static, and although largely overlooked in the plantsoil
feedback literature, the second plant that grows in the soil
will also influence the soil microbiome. Owing to the
dynamic composition of the soil microbiome, if the most
recent plant overrides the legacy of the first plant, inoculation
with specific microbiomes may only influence the early
growth phase of the second plant. However, two recent stud-
ies with a wild plant species show that plant-specific legacy
effects may persist even after another plant has grown in the
soil (Bezemer et al., 2018; Wubs & Bezemer, 2018). If this
is true, a single inoculation may influence plant growth for a
much longer time, and this could be particularly important
in crops that are grown repeatedly in the same soil.
The main goal of this study was to assess how inoculation
with soil from grass and forb species alters soil microbiomes
after growth of a second, focal plant, and whether soil inocu-
lation alters resistance to above-ground herbivorous pests in a
crop system. Based on a previous screening of the effective-
ness of soil inoculation with 37 wild plant species (Ma et al.,
2017), we selected eight species (four grasses and four forbs)
that previously improved the performance of chrysanthemum,
to generate soil inocula with distinct microbiomes. We then
inoculated sterilized soil with these species-specific soils to
assess chrysanthemum growth and we determined the compo-
sition of the soil microbiome in the inocula and in soil of
chrysanthemum after this plant had been grown in the inocu-
lated soils. Finally, we determined how inoculation influenced
the resistance of chrysanthemum to two species of above-
ground herbivorous pests, thrips and mites. These are cell-
feeding herbivores and we hypothesized that inoculation
would increase resistance against these pests via positive
plantmicrobe interactions. To understand phytochemical
mechanisms underlying induced resistance effects, we mea-
sured foliar concentrations of chlorogenic acid, which is
known to confer resistance to thrips (Leiss et al., 2009), as
well as to increase after the plant interaction with beneficial
microbes (Sanchez-Bel et al., 2016).
Specifically, we asked: does inoculation with soil from different
plant species and functional groups lead to different microbiomes
in the soil after the same crop has grown in all inoculated soils;
does soil inoculation alter plant growth and induce resistance
against herbivorous pests; and which soil microbial groups in the
inoculum and the crop microbiome correlate with plant growth
and resistance to above-ground herbivores?
Materials and Methods
Plants and herbivores
The focal plant in our study is Dendranthema 9grandiflora
(Ramat.) Kitam. cv Amadea (chrysanthemum, syn.
Chrysanthemum 9morifolium (Ramat.) Hemsl., Asteraceae).
Chrysanthemum cuttings were provided by the breeding company
Deliflor (Maasdijk, the Netherlands). Chrysanthemum is one of
the major cut flower crops worldwide and is commonly cultivated
in soil in glasshouses, which is sterilized regularly (after three to five
growth cycles, roughly once per year) to control soil pathogens (Li
et al., 2017). A culture of the thrips Frankliniella occidentalis was
established on pods of Romano beans (Vicia faba)withastarting
colony provided by the company Hazera Seeds (Made, the Nether-
lands). A culture of the spider mite Tetranychus urticae (line Sand-
poort-2) was established (Liu et al., 2017) with a starting colony
kindly provided by the group of M. Kant (University of Amster-
dam), and these were reared on detached leaves of Lima bean plants
(Phaseolus vulgaris cv Speedy). More details are provided in the
Supporting Information Methods S1.
To create different soil inocula, we selected eight wild plant
species that are typical of natural grasslands in the Netherlands
based on previous work where they exhibited positive plantsoil
feedback effects on chrysanthemum growth (Ma et al., 2017).
The species belong to two different functional groups: grasses
(Holcus lanatus (HL), Lolium perenne (LP), Alopecurus pratensis
(AP), Festuca ovina (FO)) and forbs (Achillea millefolium (AM),
Tripleurospermum maritimum (TM), Rumex acetosella (RA),
Galium mollugo (GM)).
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Soil conditioning, inoculation and plant growth
Two experiments were conducted in the experimental
glasshouse facility at the NIOO-KNAW (Wageningen, the
Netherlands). Both experiments had two phases (see further
details in Methods S2) a common procedure in plantsoil
feedback studies. In the first phase, the conditioning phase,
we grew over 2 months the eight plant species in monocul-
tures in a living soil collected from a natural grassland to cre-
ate species-specific soil inocula. In the second phase, the test
phase, we inoculated sterilized soil with 10% soil inoculum
(100 g per pot) conditioned by the different plant species
(100% sterilized soil as control) and grew chrysanthemum
plants in these soils. With this method the loss of rare
microbes should be minimal, as effects are often not observed
with dilutions below 100 times (whereas our soil was 10
times diluted) (Kurm et al., 2018).
Expt 1: Microbiomes in inocula vs soil, plant growth and
pest colonization
The aim of this experiment was to compare the soil micro-
biomes of the inocula with those after chrysanthemum had
grown in the inoculated soils, as well as the effect of soil
inoculation on the growth of plants and resistance to natu-
rally occurring herbivores. As the focus of this study is on
soils, and the changes therein by plant legacies, rhizosphere
soil was not analyzed, owing to the strong differences with
surrounding soil and because it was the surrounding soil that
was inoculated and not rhizosphere soil. Field soil was col-
lected and each soil replicate (the different pots during the
conditioning phase) was kept separate in this experiment
(n=16 soil/plant replicates). Before inoculation, a soil sample
was taken from the inocula (first six soil replicates) for micro-
biome analysis. Plants were grown in a glasshouse under con-
trolled conditions (70% relative humidity, 16 h 21°C:8h
16°C, light : dark) and after 3 wk plants were transferred to
a semi-open tunnel glasshouse. This tunnel has a plastic cover
and netting on the sides (with a mesh size that allows thrips
to enter the glasshouse), and no control of temperature, light
or humidity. Here, plants were arranged in a randomized
complete block design (one soil replicate per block). The
plants of the different soil treatments were randomly dis-
tributed inside each block. Plants had no physical barriers
between them, and were grown for seven more weeks in the
tunnel glasshouse. At the end of the experiment we quantified
the density of thrips present on each plant and at each plant
height, after which above- and below-ground biomass was
clipped and dried at 60°C and DW was determined. During
the course of the experiment, we did not observe spatial pat-
terns (i.e. differences in interior and outside plants) in thrips
colonization or damage patterns (no data available). Bulk soil
was sampled from a subset of six replicate pots per treatment
and a subsample from each pot was stored in 2 ml Eppendorf
tubes at 80°C to assess the microbiomes in the inoculated
soils after chrysanthemum had been grown in the soils.
Expt 2: Plant and herbivore performance and soil
microbiomes
After conditioning the soils, they nwere homogenized per condi-
tioning species and inoculated as described earlier. Ten replicate
pots were filled for each of the eight soil inocula and there were
10 pots filled with 100% sterilized soil. After 4 wk of chrysanthe-
mum growth, plant height was measured and three leaves were
sampled per plant to evaluate herbivore performance and plant
chemistry (see a further description later). At the end of the
experiment, the bulk soil of eight pots per soil type was collected
and a subsample from each pot was stored in 2 ml Eppendorf
tubes at 80°C for later microbiome analysis.
Thrips and mite performance Petri dish plates were prepared
with 2 ml of plant agar (1.5%) on one side of the dish. After col-
lecting the leaves, one leaf was placed in each Petri dish, with the
petiole inserted into the agar to avoid leaf desiccation, a method
shown to be effective to assess resistance to thrips (Maharijaya
et al., 2015). For each plant, the second fully mature leaf count-
ing from the top of the stem was selected to assess thrips perfor-
mance, whereas the fourth leaf was selected for spider mite
performance. Five thrips larvae (2 d old) were placed on each leaf,
and the dishes were sealed with Parafilm and placed in the same
climate chambers in which the herbivore was reared. Six days
later the number of larvae that reached prepupal stage and total
survival were recorded. To evaluate spider mite performance, one
female mite was introduced to each Petri dish, and the number of
eggs laid by this female was recorded 4 d later.
Leaf chlorogenic acid and phenolic acids The third fully
opened leaf was collected from each plant, freeze-dried and finely
ground. Ten milligrams of ground leaf material was then used in
a methanol extraction (see Methods S3). In each sample the con-
centration of chlorogenic acid and of 10 other (unidentified) phe-
nolic compounds was detected using high-performance liquid
chromatography with UV diode array detection (Olszewska,
2007), and quantified based on a chlorogenic acid standard curve
(expressed as g
1
leaf DW).
Microbiome analysis
Soil DNA was extracted from the soil samples using the
PowerSoil
®
DNA Isolation Kit according to the manufacturer’s
instructions (MoBio, Carlsbad, CA, USA). The fungal ITS2
region was amplified using the primers ITS4 and ITS9 (Ihrmark
et al., 2012) and the bacterial V4 region was targeted using the
primers 515F and 806R (Caporaso et al., 2012). Amplicons were
sequenced on the Illumina MISEQ platform (250 bp paired-end).
Both library preparation and sequencing were done at McGill
University and Genome Quebec, Canada.
Fungal sequences were analyzed using the PIPITS pipeline
(Gweon et al., 2015). FUNGUILD was used to estimate the func-
tions of fungal operational taxonomic unit (OTUs) and the out-
put of this file was compared (curated) against an in-house
database on fungal functions (Nguyen et al., 2016; Hannula
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et al., 2017). Bacterial sequences were analyzed using the Hydra
pipeline (de Hollander, 2017).
Statistical analysis
Sequencing data were normalized using the total sum scaling
(TSS) (package PHYLOSEQ in R) and OTUs occurring in less than
three samples with relative abundances of <0.01% were
removed. Furthermore, samples with <1000 or >50 000 reads
were removed from the dataset. Effects of plant species identity
and plant functional group on the structure of the bacterial and
fungal community were then examined using PERMANOVA
based on a BrayCurtis dissimilarity matrix in R (package VEGAN)
separately for both experiments. Separations among treatments
were visualized using nonmetric multidimensional scaling of a
BrayCurtis dissimilarity matrix. The Chao1 Richness index was
calculated for each sample and the effects of inoculation, soil
inocula and plant functional group on richness were evaluated
using linear models in R (mixed models for inoculation and func-
tional group with soil as random factor as described in the follow-
ing). The relative abundances were calculated as the number of
reads of an OTU, class or phylum divided by the total number of
reads in that sample. Pearson correlations were calculated
between plant and herbivore parameters, and relative abundances
of bacterial phyla and fungal classes using Bonferroni correction
for false discovery rate (package CORRPLOT in R).
The design of the experiment with different inocula and one
set of control plants (uninoculated plants) prohibits all questions
being answered with one statistical model. Therefore, plant and
herbivore data were analyzed in three steps (with different mod-
els). In the first step, we assessed the effect of inoculation per se as
the overall effect. For this, data were analyzed with mixed mod-
els, with inoculation as a fixed factor and the different soil inocula
as random factors. In the second step, we tested differences
between the soil treatments. In this analysis, soil inocula
(eight inocula plus sterilized soil) were included as fixed factors.
Whether each soil inoculum differed from sterilized soil was
tested with post hoc Dunnett tests using the ‘glht’ function of the
MULTCOMP package in R. In the third step, we assessed whether
there were differences between inocula originating from grasses
and forbs. For this last step we excluded the sterilized soil treat-
ment from the analysis. We used a mixed model with functional
group as a fixed factor and soil inocula as a random factor. For
Expt 2, in two Petri dishes, six thrips were discovered while only
five were introduced (one in a replicate from GM and one in a
replicate from LP). These data were excluded from analysis. For
the mite bioassay, those plates where no eggs were oviposited
were removed because of probable misidentification of females or
mortality of females. This resulted in four to 10 replicates per
treatment for the mite bioassay.
Data on plant height, biomass and chlorogenic acid were log-
transformed and analyzed with linear models, either with the ‘lm’
or the ‘lme’ (when random factors) function of the NLME package.
Data on the proportion of thrips reaching the prepupal stage
were analyzed using a generalized linear model with binomial dis-
tribution using the functions ‘glm’ or ‘glmer’ (when random
factors) of the LME4 package. Data on counts of mite eggs or
thrips were overdispersed and therefore analyzed with a general-
ized linear model with quasi-Poisson distributions using the func-
tions ‘glm’ or ‘glmmadmb’ (when random factors) from the
package GLMMADMB. Data from Expt 1 were all analyzed with
mixed models where block was set as random factor (in addition
to other random factors, as described earlier). All analyses were
performed in R v.3.3.3 (R Core Team, 2017).
Data availability
Paired-end DNA sequencing reads for this project have been
deposited in the European Nucleotide Archive under accession
number PRJEB35722 (http://www.ebi.ac.uk/ena/data/view/
PRJEB35722). Plant, herbivore and soil chemistry data that sup-
port the findings of this study are openly available in Datavers at
https://hdl.handle.net/10411/OIQCZH.
Results
Microbiome composition from inocula and chrysanthemum
soil
Our first aim was to assess how the bacterial and fungal commu-
nities in the soil change compared with the inocula, once
chrysanthemum grew in that soil. For this we sequenced soils
from both inocula and chrysanthemum soil from Expt 1. We
detected that the community composition for both bacteria and
fungi was most affected by whether they were from inocula or
chrysanthemum soil, with stronger differences for bacteria than
for fungi (PERMANOVA for fungi, F=10.27, R
2
=0.15,
P<0.001; for bacteria, F=63.79, R
2
=0.42, P<0.001; Fig. 1).
Furthermore, uninoculated sterilized soils differed greatly from
the inoculated soils in terms of microbial communities and were
excluded from the multivariate analysis of the soils (Fig 1). For
bacteria, the plant species and functional group had a significant
effect on the community composition in both the inocula
(PERMANOVA; plant species, F=1.32, R
2
=0.27, P<0.001;
functional group, F=1.77, R
2
=0.05, P<0.001) and in the soils
after chrysanthemum had grown in them (plant species, F=1.65,
R
2
=0.22, P<0.001; functional group, F=1.69, R
2
=0.04,
P=0.023; Fig. 1). By contrast, fungal communities were not
affected by plant species or functional group in the inoculum
(PERMANOVA; plant species, F=0.99, R
2
=0.29, P=0.52;
functional group, F=1.12, R
2
=0.05, P=0.27) or in the soils
(plant species, F=0.66, R
2
=0.19, P=0.96; functional group,
F=1.07, R
2
=0.04, P=0.37). Analyses of microbial richness in
inocula and chrysanthemum soils, as well as a description of com-
munity composition and richness from the second experiment
are shown in Notes S1 and Figs S1, S2.
Chrysanthemum effect on bacterial and fungal OTUs
We further investigated the shared proportion of OTUs between
inocula and chrysanthemum soils from Expt 1. For bacteria there
was a reduction from 5000 OTUs in the inocula to c. 4200
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OTUs in the soil, and for fungi a reduction from 220 to 110
phylotypes. For bacteria, 92% of the total OTUs were found
both in the inocula and in the soil after growth of chrysanthe-
mum (Fig. 2a), whereas only 3% of the OTUs were present in
the inoculum and these were not found later in the soil. For fungi
the situation was quite different, as only 58% of total phylotypes
were shared between the inocula and soils and 36% of the phylo-
types were lost (only present in the inocula). After chrysanthe-
mum growth, 5% of total bacterial OTUs and 8% of fungal
phylotypes were detected that were not present in the inocula.
Remarkably, out of 55 phylotypes belonging to the phylum
Mucoromycota (including the subphylum Glomeromycotina
with arbuscular mycorrhizal fungi) present in the inocula, only
four were detected in the soils after chrysanthemum growth (Figs
2b, S3). For bacteria, almost none of the OTUs were unique for
a single inoculum (Fig. 2c), while for fungi, an average of c. 8%
of the phylotypes detected in each inoculum type were unique.
However, this uniqueness in fungi in the inocula is almost lost
after chrysanthemum growth (Fig. 2c). Visualizing the similarity
between all inocula and the soils after chrysanthemum growth
reveals that for fungi not many phylotypes are shared between
inocula and soils (Fig. 2d), with the exception of LP inoculum,
which contained a high number fungal phylotypes that were
shared especially with soils inoculated with AM, AP, HL and
FO. For bacteria, all inocula shared a high similarity in terms of
shared OTUs with the soil where the same inoculum was
(a)
(b)
Fig. 1 Community structure for bacteria (a)
and fungi (b) in the inocula and in the
inoculated soils after chrysanthemum
growth, colored by plant species (sterilized
control in black) in Expt 1. Centroids are
shown as large dots and lines connecting the
individual samples to the centroids. Inocula
were conditioned by grasses (AP, Alopecurus
pratensis; FO, Festuca ovina; HL, Holcus
lanatus; LP, Lolium perenne) or forbs (AM,
Achillea millefolium; GM, Galium mollugo;
RA, Rumex acetosella; TM,
Tripleurospermum maritimum).
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introduced. RA and FO soils shared the most OTUs with their
respective inocula.
Soil inoculation effects on plant growth, resistance to
herbivores and plant defenses
In Expt 1, conducted in a semi-open tunnel glasshouse where
thrips were naturally present, chrysanthemum height was not
affected by soil inoculation overall (Fig. 3a). However, there were
differences between specific soil inocula, with plants growing in
soil conditioned by the grass AP being smaller than plants grown
in sterilized soil. Total chrysanthemum biomass showed a similar
pattern to plant height, but in this case also plants growing in soil
conditioned by the grass LP were smaller than plants growing in
100% sterilized soil (Table 1; Fig. S4). There were no significant
plant functional group effects on plant height. The number of
thrips on chrysanthemum plants was strongly reduced by soil
inoculation (Fig. 3b). Fewer thrips were observed on plants grow-
ing in soil conditioned by the grass AP and the forb RA than on
plants growing in sterilized soil. The functional group of the
plants that conditioned the inocula, however, did not affect the
number of thrips on chrysanthemum.
Fungal richness was not related to plant height (Table 2) in
Expt 1. By contrast, bacterial richness in chrysanthemum soil
explained 17% of the variation of plant height, whereas bacterial
richness in the inocula did not significantly explain plant height.
Additionally, this positive correlation was driven by those soils
containing forb inocula where bacterial richness explained 37%
of the variation in plant height, whereas in grass-inoculated soil,
bacterial richness was not correlated with plant performance. The
relationship between the number of thrips and the soil micro-
biome could not be analyzed for Expt 1 as a result of very low
numbers of thrips on the subset of plants that were included for
microbiome analysis.
In Expt 2, in a more controlled environment and with
detached-leaf assays, the percentage of thrips reaching the pupal
stage was reduced both by soil inoculation overall and by specific
soil inocula, but was not affected by the functional group (grasses
or forbs) of the conditioning plants. The percentage of thrips
reaching the pupal stage was significantly lower on plants grow-
ing in soil conditioned by the grasses HL and FO compared with
sterilized soil (Fig. 4b). Final thrips survival followed a similar
trend as the number of thrips reaching the pupal stage, although
in this case the effects of soil inoculation and specific soil inocula
were not statistically significant (P=0.08 and P=0.055, respec-
tively; Table 1; Fig. S5). In contrast to the effects on thrips, soil
inoculation, specific soil inocula and functional group did not
affect the performance of spider mites, measured as the number
of eggs laid in a period of 4 d, even though egg production was
generally lower on plants growing in inoculated than in sterilized
soil (Fig. 4c). In Expt 2, the overall effect of soil inoculation,
including the eight species-specific inocula, or the functional
group of the conditioning plants did not affect chrysanthemum
height or foliar concentrations of chlorogenic acid (Fig. 4). How-
ever, when testing the effects of species-specific inocula, chloro-
genic acid concentrations were higher in plants that grew in
inoculated soil conditioned by FO (and plant height was the low-
est) and AM than in plants growing in sterilized soil (further
results on effects of species-specific inocula are described in Notes
S2; Fig. S6). We also found that the concentrations of chloro-
genic acid (and total phenolics) were negatively correlated with
thrips performance (thrips reaching the pupal stage, R=0.42,
P=0.001; final thrips survival, R=0.32, P=0.018; Fig. 5).
Correlations of microbial groups with plant performance
and resistance to herbivores
Last, we explored the relationship between relative abundances of
bacterial and fungal taxa in the soil and plant performance and
resistance parameters from the second experiment. Plant height
was negatively correlated with eight bacterial phyla (strongest for
FCPU426 and Fibrobacteres, and one taxa therein; Fig. S7) and
with unclassified Ascomycota (Fig. 5). Only one fungal class was
positively correlated with plant height (unknown Chytrid-
iomycetes). Chlorogenic acid and total content of phenolic acids
were treated together, as the two parameters were strongly corre-
lated (r=0.85, P<0.001). Most bacterial phyla (20 out of 28
bacterial phyla) were positively correlated with the chlorogenic
acid content of the plant while only three bacterial phyla were
negatively correlated with the chlorogenic acid content (Fig. 5).
The relative abundance of three fungal classes was negatively cor-
related with chlorogenic acid and the relative abundance of two
fungal classes was positively correlated with chlorogenic acid.
These correlations were, however, all weaker than those for
detected for bacteria.
The relative abundance of 17 bacterial phyla was significantly
negatively correlated with thrips survival and the number of
pupae, while two phyla were positively correlated with thrips sur-
vival (Fig. 5). The bacterial groups with strongest correlation
were Chloroflexi, Fibrobacteres, FCPU426, Nitrospirae, Plancto-
mycetes and Saccharibacteria (Fig. S7a). From 22 fungal classes
analyzed, five classes were negatively correlated and four were
positively correlated with number of surviving thrips. Most
strongly, the relative abundance of unclassified Ascomycota and
especially members of the class Dothideomycetes were associated
with a decrease in numbers of thrips (Fig. S7b). Thrips pupation
was positively correlated with plant height but negatively corre-
lated with chlorogenic acid and total phenolics. At the microbial
level, thrips pupation was negatively correlated with bacterial
richness and, to a lesser extent, with fungal richness. Chlorogenic
acid, by contrast, was positively correlated with bacterial richness
but not with fungal richness. Most bacterial phyla (and only two
fungal classes) correlated negatively with thrips and positively
with chlorogenic acid concentrations. The number of mite eggs
on the leaves was significantly positively correlated with the rela-
tive abundance of 17 bacterial phyla and one fungal class, while
the relative abundance of three bacterial phyla and four fungal
classes were negatively correlated with the number of eggs
(Fig. 5). The strongest positive correlations were detected for Par-
cubacteria and Verrucomicrobia. The survival of mites was posi-
tively related to six bacterial phyla (with the strongest correlation
with BRC1) and negatively to one fungal class. Relative
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(a)
(b)
(c)
(d)
Fig. 2 Bacterial (left side) and fungal (right side) operational taxonomic units (OTUs) shared among inocula and inoculated soils after chrysanthemum
growth. (a) Venn diagrams of OTUs found (%) in the overall soil inocula or after chrysanthemum growth. (b) Total number of unique and shared OTUs of
inocula and soil after chrysanthemum growth, depicted at phylum/class level. (c) Venn diagrams showing total numbers of OTUs found in the different soil
inocula (oval sides) or after chrysanthemum growth (round center). The number of unique OTUs is shown in parentheses. (d) Shared OTUs between plant
species in inocula and soils, clustered by similarity. Inocula were conditioned by grasses (AP, Alopecurus pratensis; FO, Festuca ovina; HL, Holcus lanatus;
LP, Lolium perenne) or forbs (AM, Achillea millefolium; GM, Galium mollugo; RA, Rumex acetosella; TM, Tripleurospermum maritimum).
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abundances of the taxonomic groups reported in Fig. 5 in each
sample are shown in Table S1 and correlations between microbial
groups are shown in Fig. S8. A more thorough analysis of the
microbial groups aiming at genus level showing the strongest cor-
relations is shown in Fig. S9, and the average relative abundances
at this level are shown in Table S2. A full description of the rich-
ness and community composition of the soil microbiomes from
Expt 2 is also presented in Notes S3, Figs S10S12 and Table S3.
Discussion
The overall goal of this study was to assess whether applying the
concept of plantsoil feedbacks could change the soil microbiome
to a state that induces resistance to above-ground herbivorous
pests in a horticultural crop. Here, we show that soil inoculation
with species-specific conditioned soil leads to strong differences
in the microbiome in the soil in which a focal plant grows, both
in the overall community composition and in the abundance of
specific microbial groups. The functional group of the plant
species that conditioned the soil used as inoculum did not affect
chrysanthemum growth, resistance to herbivores, or different
aspects of the fungal communities in our study. However, it
influenced bacterial community assembly both in soils after
chrysanthemum growth, and in the original inocula, and it influ-
enced the relationship between bacterial richness and chrysanthe-
mum growth, and also how chrysanthemum affected soil
(a) (b)
Fig. 3 (a, b) Effects of soil inoculation on chrysanthemum height (a) and thrips numbers (b) in Expt 1 with full plants in a semi-open glasshouse with natural
thrips colonization. Sterilized soil was inoculated with 10% sterile soil or soil conditioned by grasses (AP, Alopecurus pratensis; FO, Festuca ovina; HL,
Holcus lanatus; LP, Lolium perenne) or forbs (AM, Achillea millefolium; GM, Galium mollugo; RA, Rumex acetosella; TM, Tripleurospermum
maritimum). Bars represent means SE (n= 16 plants). These parameters are analyzed for the effect of overall inoculation (inoculated or sterilized),
specific soil inocula (eight conditioned soils plus sterilized), and functional group (grasses or forbs, excluding sterilized); asterisks above bars indicate
significant differences with the sterilized soil (Dunnett test). ***, P<0.001; **, P<0.01; *, P<0.05; ns, not significant.
Table 1 Results of the statistical analyses of the plant and herbivore parameters.
Inoculation Soil inocula Functional group
F/v
21
df1; df2
2
PF/v
2
df1; df2 PF/v
2
df1; df2
2
P
Expt 1: outdoor
Thrips 5.29 1 0.02 70.58 8 <0.001 1.29 1 0.257
Total biomass 1.25 1; 7 0.301 5.53 8; 120 <0.001 1.46 1; 6 0.273
Height 0.69 1; 7 0.432 4.19 8; 120 <0.001 2.32 1; 6 0.178
Expt 2: detached leaf assays
Height 0.223 1; 7 0.651 3.05 8; 81 0.005 0.66 1; 6 0.449
Thrips pupation 4.68 1 0.030 20.12 8; 79 0.009 2.65 1 0.104
Thrips survival 3.06 1 0.080 15.21 8; 79 0.055 2.61 1 0.106
Spider mite eggs 1.23 1 0.268 40.6 8; 52 0.816 0.08 1 0.783
Chlorogenic acid 2.95 1; 7 0.129 3.10 8; 80 0.004 0.50 1; 6 0.505
Phenolics 1.21 1; 7 0.309 3.42 8; 80 0.002 1.79 1; 6 0.229
1
F-values are given for linear models; whereas v
2
are given for generalized linear models.
2
When a generalized linear mixed model was used, no residual df is given because of computational issues (Skaug et al., 2013).
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bacterial richness. We show that inoculation with soils in which
previously wild grasses and forbs were growing can induce resis-
tance in above-ground plant tissues to thrips, but not mites, both
in detached leaf assays and in semi-field conditions. We further
found a correlation of thrips resistance and of chlorogenic acid
with bacterial and fungal richness. However, although the effect
of overall inoculation consistently induced resistance, our study
also shows that the effect that a specific plant species has on
chrysanthemum growth and thrips performance via changes in
the soil inoculum is variable. This is not surprising when consid-
ering the diversity of strains, and therefore functions present in
the soil, and the interactions between them.
We hypothesized that every plant species would exert a specific
effect on the microbiome in their soil. An important question is
Table 2 Results of Pearson correlations between chrysanthemum height
from Expt 1 and bacterial and fungal richness (Chao1) in inocula
conditioned by grasses or forbs, and their respective inoculated soil after
chrysanthemum growth.
Bacteria Fungi
R
2
PR
2
P
Grass inocula 0.11 0.23 0.11 0.36
Forb inocula 0.06 0.35 0.01 0.77
Total inocula 0.01 0.65 0.02 0.57
Grass soil 0.10 0.26 0.16 0.24
Forb soil 0.37 0.008** 0.11 0.16
Total soil 0.17 0.02* 0.04 0.37
Asterisks highlight significant correlations: **, P<0.01; *, P<0.05.
(a) (b)
(c) (d)
g–1
Fig. 4 (ad) Effects of soil inoculation on chrysanthemum height (a); thrips (b) and spider mite (c) performance; and chlorogenic acid concentrations in
plants (d) in Expt 2. Sterilized soil was inoculated with 10% sterile soil or soil conditioned by grasses (AP, Alopecurus pratensis; FO, Festuca ovina; HL,
Holcus lanatus; LP, Lolium perenne) or forbs (AM, Achillea millefolium; GM, Galium mollugo; RA, Rumex acetosella; TM, Tripleurospermum
maritimum). Plants were grown in a glasshouse and herbivore performance was assessed in detached leaf assays. Bars represent means SE (n= 10 plants;
panel (c): four to 10 replicates; estimated means for generalized linear models). These parameters are analyzed for the effect of overall inoculation
(inoculated or sterilized), specific soil inocula (eight conditioned soils plus sterilized), and functional group (grasses or forbs, excluding sterilized); asterisks
above bars indicate significant differences with the sterilized soil (Dunnett test). ***, P<0.001; **, P<0.01; *, P<0.05; ns, not significant.
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how this is maintained when another plant species grows in that
soil later, as the latter plant also influences the soil microbiome
(Ma et al., 2018; Wubs & Bezemer, 2018). Here we show that
chrysanthemum exerted a strong negative effect on fungi (espe-
cially on the Glomeromycotina, known as arbuscular
mycorrhiza), as only 58% of the total fungal OTUs were present
both in the inocula and in the soil after chrysanthemum growth
(Fig. 2b). Although there were clear differences among the bacte-
rial communities of the different inocula, among the inocula and
the inoculated soils after chrysanthemum growth, between grasses
Fig. 5 Correlations between parameters of
plant performance and resistance and
relative abundance of fungal and bacterial
taxa in the soil after chrysanthemum growth
from Expt 2. The scale color of the filled
squares indicates the strength of the linear
Pearson correlation coefficients (r) and
whether it is negative (red) or positive (blue).
Only significant correlations with P<0.05
after Bonferroni correction are shown. If the
correlation is not significant, the box is left
white.
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and forb inocula, these were more resilient to the effect of
chrysanthemum growth, with >90% of bacterial OTUs shared
between inocula and inoculated soil in which chrysanthemum
had grown (Fig. 2). The effect of chrysanthemum on mycorrhizal
fungi was unexpected, as previous studies have shown coloniza-
tion by mycorrhiza of other cultivars of chrysanthemum (Wang
et al., 2018). However, later work by our group supported our
findings after observing <1% mycorrhizal root colonization with
staining techniques (H-K. Ma et al., unpublished). Recent studies
have shown that the genetic background of the test plant can
determine the root-associated microbiome and whether conspeci-
fic plantsoil feedback effects on plant growth are negative or
neutral (Hu et al., 2018; Carrillo et al., 2019). This has been an
oft-overlooked aspect during the breeding process (Perez-
Jaramillo et al., 2016; Carrillo et al., 2019), and here we show
that a certain crop or cultivar may inhibit beneficial microbial
groups such as mycorrhiza.
Grasses and forbs shaped distinct bacterial communities in the
soil and these differences remained after chrysanthemum had
been grown in soils inoculated with those communities (Fig. 1).
Remarkably, there was an unexpected strong negative correlation
between bacterial richness in the inoculum and in the inoculated
soil after chrysanthemum growth for grass-conditioned inocula
but not for forb-conditioned ones (Fig. S2). We hypothesize that
by shaping bacterial communities in the soil, grasses may enrich
the soil with grass-associated microbes and simultaneously reduce
the richness (e.g. by competition or antibiosis) of forb-associated
microbes. This means that when a forb (chrysanthemum in this
case) grows later in that soil, a higher grass-associated richness in
the inoculum leads to a lower forb-associated richness in the soil
after the forb has grown in this soil. Supporting this hypothesis,
chrysanthemum height was strongly positively correlated with
the diversity of bacteria (but not fungi) in chrysanthemum soil;
however, this was only true for forb-conditioned inocula, and not
for grass-conditioned inocula (Table 2). Soil microbial diversity
is a main driver of plant productivity under certain scenarios
(Wagg et al., 2014; Delgado-Baquerizo et al., 2016), and we
show here that this will depend on the functional group of the
plant that previously grew in that soil. Whether these differences
between grass and forbminocula are a general pattern that differs
with the functional group of the focal plant is something that
needs more attention.
Based on studies of plant interactions with individual micro-
bial strains, plant resistance to above-ground herbivores is a trait
that is partially mediated by the plant’s symbiosis with specific
soilborne microbes, known as microbial-induced systemic resis-
tance (ISR) (Pineda et al., 2010; Jung et al., 2012; Pangesti et al.,
2017). Here we show that plant resistance to herbivores also
depends on the whole soil microbiome, which can be inoculated
to enhance such resistance (Figs 3, 4). However, we cannot
exclude the possibility that, inside this complex soil microbiome,
the observed ISR could have been triggered by a single microbial
strain. In a microbiome context, the little evidence for whole
microbiome ISR that is available so far in the literature is mostly
for leaf chewers such as caterpillars or leaf beetles (Badri et al.,
2013; Hubbard et al., 2019). Until now, it was unknown if
microbiome ISR could also be effective against cell-feeding herbi-
vores such as thrips. Despite their importance as worldwide pests,
little information is available about the potential role of soilborne
microbes in reducing thrips populations. Several studies have
shown, however, that fungal endophytes (that inhabit the soil)
reduce the performance of Thrips tabaci and can reduce virus
incidence transmitted by thrips (Muvea et al., 2014; Muvea et al.,
2018). In our study, we identified 11 bacterial phyla especially
Nitrospirae and Planctomycetes and one fungal class that were
excellent candidates of beneficial microbes, that is, their relative
abundances were negatively correlated with thrips and positively
correlated with chlorogenic acid without impacting plant growth
(Fig. 5). Although these results are correlative, this opens a new
field to explore the role of those specific taxa inside the soil
microbiome and their effects on plant defenses.
When analyzing the overall effect of inoculation, no effect was
observed on plant performance, suggesting that the effects of
inoculation on herbivores are not a result of a direct relationship
between plant growth and herbivore fitness and that they may be
related to plant defenses. Especially interesting are those inocula
that led to a reduction in thrips without affecting plant growth,
which was the case for soil inocula that consisted of RA- or HL-
conditioned soil (Figs 3, 4). We expected that, in addition to
increasing resistance, the selected species and especially grasses
would promote chrysanthemum growth, based on our previous
studies where chrysanthemum grew better in soils with those
inocula than in sterilized soils (Ma et al., 2017, 2018). A possible
explanation for the lack of plant growth promotion in inoculated
soil in this study is that even for a single microbial strain, a com-
mon duality that is observed is that the establishment of symbio-
sis has a cost for the plant that might result in reduced growth
(Morgan et al., 2005), contributing to a spectrum of positive,
neutral and negative effects of microbes. At the negative side of
this spectrum, pathogen accumulation seems a common mecha-
nism for negative plant-soil feedbacks (PSFs), especially between
plants that belong to the same species (Hu et al., 2018; Wang
et al., 2019). Here we observed a reduction in plant growth and
an increase in concentrations of chlorogenic acid in the leaves of
plants growing in soil inoculated with FO (Expt 2; Fig. 4). Addi-
tionally, we found microbial groups, especially Fibrobacteres,
FCPU426 and unclassified Ascomycota, that, besides being nega-
tively correlated with thrips and positively with chlorogenic acid,
were also negatively correlated with plant growth (Fig. 5).
Chlorogenic acid has a defensive function and it can be induced
in response to below- and above-ground pathogens (Atanasova-
Penichon et al., 2012; Ma et al., 2017), but we cannot discern
here whether the lack of plant growth promotion is a result of the
presence of pathogens or to the general costs of the symbiosis. In
a microbiome context where the plant must establish a dialogue
with a multitude of strains, it is not surprising that this cost
increases and that the benefits of the soil microbiome may only
pay off under stressful scenarios.
This idea is in line with the priming concept where the plant
defensive response is primed by microbes but then only mounted
after attack or stress. Accordingly, the benefit of the microbial
symbiosis on plant fitness is only evident in the presence of the
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attacker (van Hulten et al., 2006; Martınez-Medina et al., 2016).
Our study was not designed to assess the priming mechanism,
and plant height and chlorogenic acid were measured before her-
bivore attack. Future work with model systems for which molec-
ular tools are available could confirm the role of defensive
compounds in microbiome ISR and priming. Therefore, the
changes in chlorogenic acid might be even stronger in a real sce-
nario where plants are responding to their attackers, as has been
previously shown for plants colonized with mycorrhizas and
attacked by pathogens (Sanchez-Bel et al., 2016). Also important
is the fact that microbiomes are an intrinsic component of the
plant as a holobiont, providing plants with an extended pheno-
type (Vandenkoornhuyse et al., 2015). Inside a microbial com-
munity, where some strains might contribute to nutrient
acquisition, others may enhance tolerance to drought, and still
others resistance to pathogens. Here we isolated the effects on
herbivorous insects by performing the experiments in controlled
conditions with abundant water, nutrients and a relative absence
of pathogens. Based on this, we expect that the effects reported
could be amplified in field conditions in the presence of other
biotic and abiotic stresses, as it has also been suggested for the
general effects of PSFs (De Long et al., 2019).
In conclusion, we show that soil inoculation and the applica-
tion of plantsoil feedbacks to create different soil microbiomes
comprise a strategy that can reduce pest incidence in above-
ground tissues. Although the application of plantsoil feedbacks
for pest control via influencing the soil microbiome had been
suggested previously, empirical evidence from agricultural sys-
tems has been lacking so far. A major challenge is how to select
conditioning plants that create beneficial soil microbiomes that
consistently reduce pests and promote plant growth, within the
context of highly diverse and variable soil microbiomes. Hence,
the ‘holy grail’ in research on microbiome-induced plant resis-
tance is to find plant species that modify the soil microbiome
in a predictable and desirable way. Following a top-down
approach, the concept of plantsoil feedbacks could also be used
as a source of discovery of keystone microbial taxa that induce
resistance in plants. This study contributes to the necessary
change in the paradigm of agricultural practices, where in addi-
tion to focusing on reducing negative plantsoil feedbacks and
pathogenic microbes, more attention should be paid to making
use of positive PSFs and beneficial microbes. Ecologically based
strategies are needed to improve the sustainability of our agri-
cultural systems, and our study emphasizes that the soil is a key
component.
Acknowledgements
This work was funded by the Netherlands Organisation for Sci-
entific Research, in collaboration with Biobest Group NV, Lan-
delijke commissie (LC) Chrysant, and Wageningen University
Business Unit Glasshouse Horticulture Bleiswijk (NWO Groen,
grant no. 870.15.080 and Vici grant no. 865.14.006). IK was
funded by sabbatical grants from KNAW, NWO and The Grad-
uate School for Production Ecology & Resource Conservation.
We thank the following people for providing materials and
advise: A. Post from Deliflor for the chrysanthemum cuttings, T.
Snoeren from Hazera for the thrips to start our own colony, and
S. Legarrea and M. Kant for the mites. We also thank S. van den
Brande, Y. Li, M. van der Sluis, R. Heinen and especially H-K.
Ma for their help during the experiments. Sequencing of the sam-
ples was performed in collaboration with McGill University and
Genome Quebec Innovation Centre, Canada. We also thank the
three anonymous reviewers for their insightful comments on a
previous version.
Author contributions
AP, IK and TMB design the research; AP, IK and WG per-
formed the research and collected the data; AP, IK, SEH and
TMB analyzed and interpreted the data; AP and SEH wrote the
initial version of the manuscript and all authors contributed to its
revision.
ORCID
T. Martijn Bezemer https://orcid.org/0000-0002-2878-3479
S. Emilia Hannula https://orcid.org/0000-0003-1398-2018
Ian Kaplan https://orcid.org/0000-0003-4469-2750
Ana Pineda https://orcid.org/0000-0003-3854-5674
References
Atanasova-Penichon V, Pons S, Pinson-Gadais L, Picot A, Marchegay G,
Bonnin-Verdal M-N, Ducos C, Barreau C, Roucolle J, Sehabiague P. 2012.
Chlorogenic acid and maize ear rot resistance: a dynamic study investigating
Fusarium graminearum development, deoxynivalenol production, and phenolic
acid accumulation. Molecular PlantMicrobe Interactions 25: 16051616.
Badri DV, Zolla G, Bakker MG, Manter DK, Vivanco JM. 2013. Potential
impact of soil microbiomes on the leaf metabolome and on herbivore feeding
behavior. New Phytologist 198: 264273.
Bardgett RD, Wardle DA. 2010. Aboveground-belowground linkages: biotic
interactions, ecosystem processes, and global change. Oxford, UK: Oxford
University Press.
Bennett JA, Klironomos J. 2018. Mechanisms of plantsoil feedback:
interactions among biotic and abiotic drivers. New Phytologist 222:9196.
Bezemer TM, Jing J, Bakx-Schotman JT, Bijleveld EJ. 2018. Plant competition
alters the temporal dynamics of plant-soil feedbacks. Journal of Ecology 106:
22872300.
Bulgarelli D, Schlaeppi K, Spaepen S, Loren Ver, van Themaat E, Schulze-
Lefert P. 2013. Structure and functions of the bacterial microbiota of plants.
Annual Review of Plant Biology 64: 807838.
Busby PE, Soman C, Wagner MR, Friesen ML, Kremer J, Bennett A, Morsy M,
Eisen JA, Leach JE, Dangl JL. 2017. Research priorities for harnessing plant
microbiomes in sustainable agriculture. PLoS Biology 15: e2001793.
Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N,
Owens SM, Betley J, Fraser L, Bauer M et al. 2012. Ultra-high-throughput
microbial community analysis on the Illumina HiSeq and MiSeq platforms.
ISME Journal 6: 1621.
Carrillo J, Ingwell LL, Li X, Kaplan I. 2019. Domesticated tomatoes are more
vulnerable to negative plantsoil feedbacks than their wild relatives. Journal of
Ecology 107: 17531766.
De Long JR, Fry EL, Veen GF, Kardol P. 2019. Why are plantsoil feedbacks so
unpredictable, and what to do about it? Functional Ecology 33: 118128.
Delgado-Baquerizo M, Maestre FT, Reich PB, Jeffries TC, Gaitan JJ, Encinar
D, Berdugo M, Campbell CD, Singh BK. 2016. Microbial diversity drives
multifunctionality in terrestrial ecosystems. Nature Communications 7: 10541.
New Phytologist (2020) 226: 595–608 Ó2019 The Authors
New Phytologist Ó2019 New Phytologist Trust
www.newphytologist.com
Research
New
Phytologist
606
Gadhave KR, Hourston JE, Gange AC. 2016. Developing soil microbial
inoculants for pest management: can one have too much of a good thing?
Journal of Chemical Ecology 42: 348356.
Gweon HS, Oliver A, Taylor J, Booth T, Gibbs M, Read DS, Griffiths RI,
Schonrogge K. 2015. PIPITS: an automated pipeline for analyses of fungal
internal transcribed spacer sequences from the Illumina sequencing platform.
Methods in Ecology and Evolution 6: 973980.
Hannula SE, Morrien E, de Hollander M, Van der Putten WH, de Boer W, Van
Veen JA. 2017. Shifts in rhizosphere fungal community during secondary
succession following abandonment from agriculture. ISME J 11: 22942304.
Hannula SE, Zhu F, Heinen R, Bezemer TM. 2019. Foliar-feeding insects
acquire microbiomes from the soil rather than the host plant. Nature
Communications 10: 1254.
Heinen R, van der Sluijs M, Biere A, Harvey JA, Bezemer TM. 2018. Plant
community composition but not plant traits determine the outcome of soil
legacy effects on plants and insects. Journal of Ecology 106: 12171229.
Herrera Paredes S, Gao T, Law TF, Finkel OM, Mucyn T, Teixeira PJPL, Salas
Gonzalez I, Feltcher ME, Powers MJ, Shank EA et al. 2018. Design of
synthetic bacterial communities for predictable plant phenotypes. PLoS Biology
16: e2003962.
de Hollander M. 2017. nioo-knaw/hydra: 1.3.3. Zenodo. [WWW document]
URL doi: 10.5281/zenodo.884028.
Hu L, Robert CAM, Cadot S, Zhang X, Ye M, Li B, Manzo D, Chervet N,
Steinger T, van der Heijden MGA et al. 2018. Root exudate metabolites drive
plant-soil feedbacks on growth and defense by shaping the rhizosphere
microbiota. Nature Communications 9: 2738.
Hubbard CJ, Li B, McMinn R, Brock MT, Maignien L, Ewers BE, Kliebenstein
D, Weinig C. 2019. The effect of rhizosphere microbes outweighs host plant
genetics in reducing insect herbivory. Molecular Ecology 28: 18011811.
Ihrmark K, Bodeker ITM, Cruz-Martinez K, Friberg H, Kubartova A, Schenck
J, Strid Y, Stenlid J, Brandstrom-Durling M, Clemmensen KE et al. 2012.
New primers to amplify the fungal ITS2 region evaluation by 454-
sequencing of artificial and natural communities. FEMS Microbiology Ecology
82: 666677.
Jung S, Martinez-Medina A, Lopez-Raez J, Pozo M. 2012. Mycorrhiza-induced
resistance and priming of plant defenses. Journal of Chemical Ecology 38: 651
664.
Kaplan I, Pineda A, Bezemer M. 2018. Application and theory of plantsoil
feedbacks on aboveground herbivores. In: Ohgushi T, Wurst S, Johnson SN,
eds. Aboveground-belowground community ecology. Cham, Switzerland: Springer
International, 319343.
Kos M, Tuijl MAB, de Roo J, Mulder PPJ, Bezemer TM. 2015. Species-specific
plantsoil feedback effects on above-ground plantinsect interactions. Journal
of Ecology 103: 904914.
Kurm V, van der Putten WH, Pineda A, Hol WHG. 2018. Soil microbial
species loss affects plant biomass and survival of an introduced bacterial strain,
but not inducible plant defences. Annals of Botany 121: 311319.
Leiss KA, Maltese F, Choi YH, Verpoorte R, Klinkhamer PGL. 2009.
Identification of Chlorogenic acid as a resistance factor for thrips in
Chrysanthemum.Plant Physiology 150: 15671575.
Li P, Chen J, Li Y, Zhang K, Wang H. 2017. Possible mechanisms of control of
Fusarium wilt of cut chrysanthemum by Phanerochaete chrysosporium in
continuous cropping fields: a case study. Scientific Reports 7: 15994.
Liu J, Legarrea S, Kant MR. 2017. Tomato reproductive success is equally
affected by herbivores that induce or that suppress defenses. Frontiers in Plant
Science 8: 2128.
Ma H-k, Pineda A, van der Wurff AWG, Bezemer TM. 2018. Carry-over effects
of soil inoculation on plant growth and health under sequential exposure to
soil-borne diseases. Plant and Soil 433: 257270.
Ma H-k, Pineda A, van der Wurff AWG, Raaijmakers C, Bezemer TM. 2017.
Plantsoil feedback effects on growth, defense and susceptibility to a soil-borne
disease in a cut flower crop: species and functional group effects. Frontiers in
Plant Science 8: 2127.
Maharijaya A, Vosman B, Steenhuis-Broers G, Pelgrom K, Purwito A, Visser
RGF, Voorrips RE. 2015. QTL mapping of thrips resistance in pepper.
Theoretical and Applied Genetics 128: 19451956.
Mariotte P, Mehrabi Z, Bezemer TM, De Deyn GB, Kulmatiski A, Drigo B,
Veen GF, van der Heijden MGA, Kardol P. 2018. Plantsoil feedback:
bridging natural and agricultural sciences. Trends in Ecology & Evolution 33:
129142.
Martınez-Medina A, Flors V, Heil M, Mauch-Mani B, Pieterse CM, Pozo MJ,
Ton J, van Dam NM, Conrath U. 2016. Recognizing plant defense priming.
Trends in Plant Science 21: 818822.
Middleton EL, Bever JD. 2012. Inoculation with a native soil community
advances succession in a grassland restoration. Restoration Ecology 20: 218226.
Morgan J, Bending G, White P. 2005. Biological costs and benefits to plant
microbe interactions in the rhizosphere. Journal of Experimental Botany 56:
17291739.
Muvea AM, Meyhofer R, Subramanian S, Poehling H-M, Ekesi S, Maniania
NK. 2014. Colonization of onions by endophytic fungi and their impacts on
the biology of Thrips tabaci.PLoS ONE 9: e108242.
Muvea AM, Subramanian S, Maniania NK, Poehling H-M, Ekesi S,
Meyhoefer R. 2018. Endophytic colonization of onions induces resistance
against viruliferous thrips and virus replication. Frontiers in Plant Science
9: 1785.
Nguyen NH, Song Z, Bates ST, Branco S, Tedersoo L, Menke J, Schilling
JS, Kennedy PG. 2016. FUNGuild: an open annotation tool for parsing
fungal community datasets by ecological guild. Fungal Ecology 20: 241
248.
Olszewska M. 2007. Quantitative HPLC analysis of flavonoids and chlorogenic
acid in the leaves and inflorescences of Prunus serotina Ehrh. Acta
Chromatographica 19: 253269.
Pangesti N, Vandenbrande S, Pineda A, Dicke M, Raaijmakers JM, Van Loon J.
2017. Antagonism between two root-associated beneficial Pseudomonas strains
does not affect plant growth promotion and induced resistance against a leaf-
chewing herbivore. FEMS Microbiology Ecology 93: fix1038.
Peralta AL, Sun Y, McDaniel MD, Lennon JT. 2018. Crop rotational diversity
increases disease suppressive capacity of soil microbiomes. Ecosphere 9: e02235.
Perez-Jaramillo JE, Mendes R, Raaijmakers JM. 2016. Impact of plant
domestication on rhizosphere microbiome assembly and functions. Plant
Molecular Biology 90: 635644.
Pieterse CMJ, de Jonge R, Berendsen RL. 2016. The soil-borne supremacy.
Trends in Plant Science 21: 171173.
Pieterse CMJ, Zamioudis C, Berendsen RL, Weller DM, van Wees SCM,
Bakker PAHM. 2014. Induced systemic resistance by beneficial microbes.
Annual Review of Phytopathology 52: 347375.
Pineda A, Kaplan I, Bezemer TM. 2017. Steering soil microbiomes to suppress
aboveground insect pests. Trends in Plant Science 22: 770778.
Pineda A, Zheng S-J, van Loon JJA, Pieterse CMJ, Dicke M. 2010. Helping
plants to deal with insects: the role of beneficial soil-borne microbes. Trends in
Plant Science 15: 507514.
R Core Team. 2017. R: a language and environment for statistical computing,
v.3.3.3. Vienna, Austria: R Foundation for Statistical Computing. [WWW
document] URL https://www.R-project.org/ [accessed 24 January 2020].
Raaijmakers JM, Mazzola M. 2016. Soil immune responses. Science 352: 1392
1393.
Rashid MH-O, Chung YR. 2017. Induction of systemic resistance against insect
herbivores in plants by beneficial soil microbes. Frontiers in Plant Science 8:
1816.
Sanchez-Bel P, Troncho P, Gamir J, Pozo MJ, Cama~nes G, Cerezo M, Flors V.
2016. The nitrogen availability interferes with mycorrhiza-induced resistance
against Botrytis cinerea in tomato. Frontiers in Microbiology 7: 1598.
Santhanam R, Luu VT, Weinhold A, Goldberg J, Oh Y, Baldwin IT. 2015.
Native root-associated bacteria rescue a plant from a sudden-wilt disease that
emerged during continuous cropping. Proceedings of the National Academy of
Sciences, USA 112: E5013E5020.
Schlatter D, Kinkel L, Thomashow L, Weller D, Paulitz T. 2017. Disease
suppressive soils: new insights from the soil microbiome. Phytopathology 107:
12841297.
Skaug H, Fournier D, Nielsen A, Magnusson A, Bolker B. 2013. Generalized
linear mixed models using AD model builder, v.0.7.7. [WWW document] URL
http://glmmadmb.r-forge.r-project.org [accessed 24 January 2020].
Ó2019 The Authors
New Phytologist Ó2019 New Phytologist Trust
New Phytologist (2020) 226: 595–608
www.newphytologist.com
New
Phytologist Research 607
Timmusk S, Behers L, Muthoni J, Muraya A, Aronsson A-C. 2017. Perspectives
and challenges of microbial application for crop improvement. Frontiers in
Plant Science 8: 49.
van der Putten WH, Bardgett RD, Bever JD, Bezemer TM, Casper BB, Fukami
T, Kardol P, Klironomos JN, Kulmatiski A, Schweitzer JA et al. 2013. Plant
soil feedbacks: the past, the present and future challenges. Journal of Ecology
101: 265276.
van Hulten M, Pelser M, van Loon LC, Pieterse CMJ, Ton J. 2006. Costs and
benefits of priming for defense in Arabidopsis.Proceedings of the National
Academy of Sciences, USA 103: 56025607.
Vandenkoornhuyse P, Quaiser A, Duhamel M, Le Van A, Dufresne A. 2015.
The importance of the microbiome of the plant holobiont. New Phytologist
206: 11961206.
Wagg C, Bender SF, Widmer F, van der Heijden MG. 2014. Soil biodiversity
and soil community composition determine ecosystem multifunctionality.
Proceedings of the National Academy of Sciences, USA 111: 52665270.
Wall DH, Nielsen UN, Six J. 2015. Soil biodiversity and human health. Nature
528:6976.
Wang M, Ruan W, Kostenko O, Carvalho S, Hannula SE, Mulder PPJ, Bu F,
Putten WH, Bezemer TM. 2019. Removal of soil biota alters soil feedback
effects on plant growth and defense chemistry. New Phytologist 221: 1478
1491.
Wang Y, Wang M, Li Y, Wu A, Huang J. 2018. Effects of arbuscular
mycorrhizal fungi on growth and nitrogen uptake of Chrysanthemum
morifolium under salt stress. PLoS ONE 13: e0196408.
Wubs ERJ, Bezemer TM. 2018. Temporal carry-over effects in sequential plant
soil feedbacks. Oikos 127: 220229.
Wubs ERJ, van der Putten WH, Bosch M, Bezemer TM. 2016. Soil inoculation
steers restoration of terrestrial ecosystems. Nature Plants 2: 16107.
Yuan J, Zhao J, Wen T, Zhao M, Li R, Goossens P, Huang Q, Bai Y, Vivanco
JM, Kowalchuk GA et al. 2018. Root exudates drive the soil-borne legacy of
aboveground pathogen infection. Microbiome 6: 156.
Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 Richness of bacteria and fungal phylotypes in the inocula
(upper panels) and in the soil after chrysanthemum growth (bot-
tom panels).
Fig. S2 Correlations between the richness (Chao1 index) of bac-
teria (left) and fungi (right) in the inocula and in the soils after
chrysanthemum growth in Expt 1.
Fig. S3 Richness (Chao1) of fungal phylotypes of the subphylum
Glomeromycotina.
Fig. S4 Effects of soil inoculation on chrysanthemum biomass of
plants from Expt 1.
Fig. S5 Effects of soil inoculation on thrips survival from Expt 2.
Fig. S6 Effects of soil inoculation on Phenolics in leaves from
uninfested plants from Expt 2.
Fig. S7 The relative abundance of bacterial (a) and fungal (b)
groups showing the strongest correlation with thrips performance
in Expt 2.
Fig. S8 Correlations between parameters of plant performance
and resistance and relative abundance of fungal and bacterial taxa
in Expt 2.
Fig. S9 Correlations at the genus level between parameters of
plant performance and resistance and relative abundance of fun-
gal and bacterial taxa in Expt 2.
Fig. S10 Bacterial and fungal richness (top panels) and commu-
nity composition (bottom panels) in the soil after chrysanthe-
mum growth from Expt 2.
Fig. S11 The relative abundance of bacterial phyla that were
affected by the soil conditioning and inoculation in Expt 2.
Fig. S12 The relative abundance of fungal phyla that were
affected by the soil conditioning and inoculation in Expt 2.
Methods S1 Herbivore rearing.
Methods S2 Soil preparation and plant growth.
Methods S3 Chemical analysis of phenolics.
Notes S1 Soil inoculation effects on microbial richness in inocula
and soils.
Notes S2 Soil inoculation effects on plant growth and plant
defenses.
Notes S3 Microbial richness and community composition of the
chrysanthemum soils from Expt 2.
Table S1 Data used to make Figs 5 and S9 which show correla-
tions between parameters of plant performance and resistance
and relative abundance of fungal and bacterial taxa.
Table S2 Average relative abundances of bacteria and fungi at the
lowest taxonomic level that could be identified in chrysanthe-
mum soil and inocula.
Table S3 Effect of different factors on the relative abundances of
bacterial and fungal taxonomic groups.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
New Phytologist (2020) 226: 595–608 Ó2019 The Authors
New Phytologist Ó2019 New Phytologist Trust
www.newphytologist.com
Research
New
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Medicago sativa L. (alfalfa, syn. lucerne) is an important forage crop for livestock, which is subject to attack from a range of insect pests and susceptible to diseases that can reduce production and persistence. This review considers the main insect pests affecting M. sativa in China and New Zealand as well as the wider plant resistance mechanisms and multitrophic interaction that occur between plants, insect pests, entomopathogens, endophytes, the environment, and climate change. This is with a view to identifying new research opportunities applicable to M. sativa that can be applied to improving production and persistence of this important agricultural crop. These opportunities include identification and activity of entomopathogens/endophytes (e.g., Bacillus and Pseudomonas spp., Metarhizium spp.) and plant growth enhancers (Trichoderma), as well as multitrophic plant-insect-microbial interactions.
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In agricultural ecosystems, insect pests, pathogens, weather patterns, and reduced soil fertility pose major challenges to crop productivity and are responsible for significant yield losses worldwide. Iris yellow spot virus (IYSV) vectored by Thrips tabaci Lindeman, is a major hindrance to onion production in eastern Africa. Control measures often rely on insecticides with deleterious effects. Endophytes are one key alternative as they can play important roles in mediating induced systemic resistance. Hence, we examined the potential effect of endophytic fungus Hypocrea lixii (F3ST1) on feeding and replication of IYSV on endophyte-colonized (E+) and endophyte-free (E−) onion plants. For more precise assessment, replication was also tested using leaf disk bioassays and individual thrips. The number of feeding punctures was significantly lower in E+ as compared to E− plants. Disease level was significantly lower in E+ as compared to E− plants for four weeks post-exposure to thrips. IYSV replication was reduced by 2.5-fold in endophytic treatment on both whole plant and leaf disk assays. Thrips tabaci showed 2 times higher feeding activities on endophyte-free onion leaf disks as compared to the endophyte-inoculated leaf disks. Our results suggest potential utility of the endophytes to reduce feeding damage and virus infection on onion plants. Further studies should be conducted to elucidate the secondary metabolites involved in such endophyte-thrips-virus mediated interaction and determine whether the interactions extend for this and other onion varieties and viruses under field conditions.
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Plant–soil feedback (PSF) occurs when plants alter soil properties that influence the performance of seedlings, with consequent effects on plant populations and communities. Many processes influence PSF, including changes in nutrient availability and the accumulation of natural enemies, mutualists, or secondary chemicals. Typically, these mechanisms are investigated in isolation, yet no single mechanism is likely completely responsible for PSF as these processes can interact. Further, the outcome depends on which resources are limiting and the other plants and soil biota in the surrounding environment. As such, understanding the mechanisms of PSF and their role within plant communities requires quantifying the interactions among the processes influencing PSF and the associated abiotic and biotic contexts. This article is protected by copyright. All rights reserved.
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Background and aims Most plant-soil feedback and inoculation studies are limited to one growth cycle. We examined the effects of inoculation with eight plant-conditioned soils on chrysanthemum during two sequential growth cycles. The plants were also exposed sequentially to soil diseases. Methods In cycle 1, plants were grown in sterile soil inoculated or not with plant-conditioned soils, and exposed or not to Pythium or root feeding nematodes. In cycle 2, new plants were grown in soil from cycle 1 or in new 100% sterile soil. Plants were exposed again to Pythium, or to soil with pathogens and nematodes collected from a commercial chrysanthemum greenhouse. Results After two cycles, effects of soil inoculation on plant growth were still present. Chrysanthemum exhibited a negative conspecific feedback response, but this was less strong in inoculated soils. Pythium or nematode addition did not affect plant growth. However, addition of pathogen-containing soil from the commercial greenhouse reduced plant growth in sterile soil but increased growth in plant-conditioned soils. Conclusions Inoculation with plant-conditioned soil can reduce the negative conspecific plant-soil feedback of chrysanthemum. Our study further advances our understanding of the temporal dynamics of conspecific and heterospecific plant-soil feedbacks, and how they interact with soil-borne diseases.
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We examined how removal of soil biota affects plant–soil feedback (PSF) and defense chemistry of Jacobaea vulgaris, an outbreak plant species in Europe containing defense compounds pyrrolizidine alkaloids (PAs). Macrofauna and mesofauna, as well as fungi and bacteria were removed size‐selectively from unplanted soil, or soil with J. vulgaris exposed or not to above‐ or belowground insect herbivores. Wet‐sieved fractions, using 1000‐, 20‐, 5‐, and 0.2‐μm mesh sizes were added to sterilized soil and new plants were grown. Sieving treatments were verified by molecular analysis of the inocula. In the feedback phase, plant biomass was lowest in soils with 1000 and 20 μm inocula, and soils conditioned with plants gave more negative feedback than without plants. Remarkably, part of this negative PSF effect remained present in the 0.2 μm inoculum where no bacteria were present. PA concentration and composition of plants with 1000 or 20 μm inocula, differed from those with 5 or 0.2 μm inocula; however, only if soils had been conditioned by undamaged plants or plants damaged by aboveground herbivores. These effects correlated with leaf hyperspectral reflectance. We conclude that size‐selective removal of soil biota altered PSFs, but that these PSFs were also influenced by herbivory during the conditioning phase. This article is protected by copyright. All rights reserved.
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Background: Plants are capable of building up beneficial rhizosphere communities as is evidenced by disease-suppressive soils. However, it is not known how and why soil bacterial communities are impacted by plant exposure to foliar pathogens and if such responses might improve plant performance in the presence of the pathogen. Here, we conditioned soil by growing multiple generations (five) of Arabidopsis thaliana inoculated aboveground with Pseudomonas syringae pv tomato (Pst) in the same soil. We then examined rhizosphere communities and plant performance in a subsequent generation (sixth) grown in pathogen-conditioned versus control-conditioned soil. Moreover, we assessed the role of altered root exudation profiles in shaping the root microbiome of infected plants. Results: Plants grown in conditioned soil showed increased levels of jasmonic acid and improved disease resistance. Illumina Miseq 16S rRNA gene tag sequencing revealed that both rhizosphere and bulk soil bacterial communities were altered by Pst infection. Infected plants exhibited significantly higher exudation of amino acids, nucleotides, and long-chain organic acids (LCOAs) (C > 6) and lower exudation levels for sugars, alcohols, and short-chain organic acids (SCOAs) (C ≤ 6). Interestingly, addition of exogenous amino acids and LCOA also elicited a disease-suppressive response. Conclusion: Collectively, our data suggest that plants can recruit beneficial rhizosphere communities via modification of plant exudation patterns in response to exposure to aboveground pathogens to the benefit of subsequent plant generations.
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1.Domesticated plants can differ from their wild counterparts in the strength and outcome of species interactions, both above‐ and belowground. Plant‐soil feedbacks influence plant success, and plant‐associated soil microbial communities can influence plant interactions with herbivores and their natural enemies, yet, it is unclear if domestication has changed these relationships. 2.To determine the effects of domestication on plant‐soil interactions, we characterized soil microbial communities associated with various cultivars of domesticated tomato and some of its wild relatives. We measured the strength and direction of plant‐soil feedbacks for domesticated and wild tomatoes, and the effects of soil on plant resistance to specialist herbivory by Manduca sexta, and the attraction of a parasitoid wasp, Cotesia congregata. 3.Domesticated tomatoes and their wild relatives had negative plant‐soil feedbacks, as conspecifics cultivated soil that negatively impacted performance of subsequent plants (longer germination time, lower biomass) than if they grew in non‐tomato soils. Significant variation existed among domesticated and wild tomato varieties in the strength of these feedbacks, ranging from neutral to strongly negative. For aboveground plant biomass, tomato wild relatives were unaffected by growing in tomato‐conditioned soil while domesticated tomatoes grew smaller in tomato soil, indicating effects of plant domestication. Overall, increased microbial biomass within the rhizosphere resulted in progressively less‐negative plant‐soil feedbacks. 4.Plant cultivars had different levels of resistance to herbivory by M. sexta, but this did not depend on plant domestication or soil type. The parasitoid C. congregata was primarily attracted to herbivore damaged plants, independent of plant domestication status, and for these damaged plants, wasps preferred some cultivars over others, and wild plants grown in tomato soil over wild plants grown in non‐tomato soil. 5.Synthesis: These results indicate that crop tomatoes are more likely to show negative plant‐soil feedbacks than wild progenitors, which could partially explain their sensitivity to monocultures in agricultural soils. Further, cultivar‐specific variation in the ability to generate soil microbial biomass, independent of domestication status, appears to buffer the negative consequences of sharing the same soil. Last, soil legacies were relatively absent for herbivores, but not for parasitoid wasps, suggesting trophic level specificity in soil feedbacks on plant‐insect interactions. This article is protected by copyright. All rights reserved.
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Rhizosphere microbes affect plant performance, including plant resistance against insect herbivores; yet, a direct comparison of the relative influence of rhizosphere microbes vs. plant genetics on herbivory levels and on metabolites related to defense is lacking. In the crucifer Boechera stricta, we tested the effects of rhizosphere microbes and plant population on herbivore resistance, the primary metabolome, and select secondary metabolites. Plant populations differed significantly in the concentrations of 6 glucosinolates (GLS), secondary metabolites known to provide herbivore resistance in the Brassicaceae. The population with lower GLS levels experienced ~60% higher levels of aphid (Myzus persicae) attack; no association was observed between GLS and damage by a second herbivore, flea beetles (Phyllotreta cruciferae). Rhizosphere microbiome (disrupted vs. intact native microbiome) had no effect on plant GLS concentrations. However, aphid number and flea beetle damage were respectively ~3‐fold and 7‐fold higher among plants grown in the disrupted vs. intact native microbiome treatment. These differences may be attributable to shifts in primary metabolic pathways previously implicated in host defense against herbivores, including increases in pentose and glucoronate interconversion among plants grown with an intact microbiome. Further, native microbiomes with distinct community composition (as estimated from 16s rRNA amplicon sequencing) differed 2‐fold in their effect on host plant susceptibility to aphids. The findings suggest that rhizosphere microbes, including distinct native microbiomes, can play a greater role than population in defense against insect herbivores, and act through metabolic mechanisms independent of population. This article is protected by copyright. All rights reserved.
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1.The study of feedbacks between plants and soils (plant‐soil feedbacks; PSFs) is receiving increased attention. However, PSFs have been mostly studied in isolation of abiotic and biotic drivers that could affect their strength and direction. This is problematic because it has led to limited predictive power of PSFs in ‘the real world’, leaving large knowledge gaps in our ability to predict how PSFs contribute to ecosystem processes and functions. 2.Here, we present a synthetic framework to elucidate how abiotic and biotic drivers affect PSFs. We focus on two key abiotic drivers (temperature and soil moisture) and two key biotic drivers (aboveground plant consumers and belowground top‐down control of pathogens and mutualists). We focus on these factors because they are known drivers of plants and soil organisms and the ecosystem processes they control, and hence would be expected to strongly influence PSFs. 3.Our framework describes the proposed mechanisms behind these drivers and explores their effects on PSFs. We demonstrate the impacts of these drivers using the fast‐ to slow‐growing plant economics spectrum. We use this well‐established paradigm because plants on opposite ends of this spectrum differ in their relationships with soil biota and have developed contrasting strategies to cope with abiotic and biotic environmental conditions. 4.Finally, we present suggestions for improved experimental designs and scientific inference that will capture and elucidate the influence of above‐ and belowground drivers on PSFs. By establishing the role of abiotic and biotic drivers of PSFs, we will be able to make more robust predictions of how PSFs impact on ecosystem function. This article is protected by copyright. All rights reserved.
Chapter
Plant–soil feedbacks are legacy effects created by an initial plant on the growth of subsequent plants using the same soil. These indirect soil-mediated interactions are primarily studied in the context of changes in the belowground biotic community. Here, we review current evidence surrounding plant–soil feedbacks, focusing on how these interactions are studied from an experimental standpoint and expand this discussion into new directions surrounding the influence of feedbacks on interactions with aboveground herbivorous insects. The taxon-specific impact of individual soil groups on foliar-feeding insects is well-described, but expanding this framework to plant–soil feedbacks is challenging because different plant species cause simultaneous and dramatic shifts in the composition of all soil life, sometimes in contradictory directions (i.e., certain fungi may increase, while nematodes decrease). Thus, expanding simple pair-wise root herbivore–plant–insect relationships to more holistic approaches that account for the full spectrum of changes in soil biota represents both a mechanistic and analytical challenge. These community-wide shifts, however, are representative of true legacy effects encountered by plants and insects in nature. We end our chapter on a discussion of how plant–soil feedbacks can be functionally used to steer the microbiome for enhanced crop protection in applied agricultural systems.