Exposure to the leaf litter microbiome of healthy adults protects seedlings from pathogen damage

Abstract and Figures

It is increasingly recognized that microbiota affect host health and physiology. However, it is unclear what factors shape microbiome community assembly in nature, and how microbiome assembly can be manipulated to improve host health. All plant leaves host foliar endophytic fungi, which make up a diverse, environmentally acquired fungal microbiota. Here, we experimentally manipulated assembly of the cacao tree (Theobroma cacao) fungal microbiome in nature and tested the effect of assembly outcome on host health. Using next-generation sequencing, as well as culture-based methods coupled with Sanger sequencing, we found that manipulating leaf litter exposure and location within the forest canopy significantly altered microbiome composition in cacao. Exposing cacao seedlings to leaf litter from healthy conspecific adults enriched the seedling microbiome with Colletotrichum tropicale, a fungal endophyte known to enhance pathogen resistance of cacao seedlings by upregulating host defensive pathways. As a result, seedlings exposed to healthy conspecific litter experienced reduced pathogen damage. Our results link processes that affect the assembly and composition of microbiome communities to their functional consequences for host success, and have broad implications for understanding plant–microbe interactions. Deliberate manipulation of the plant–fungal microbiome also has potentially important applications for cacao production and other agricultural systems in general. © 2017 The Author(s) Published by the Royal Society. All rights reserved.
Content may be subject to copyright.
Cite this article: Christian N, Herre EA, Mejia
LC, Clay K. 2017 Exposure to the leaf litter
microbiome of healthy adults protects
seedlings from pathogen damage. Proc. R. Soc.
B284: 20170641.
Received: 26 March 2017
Accepted: 30 May 2017
Subject Category:
Subject Areas:
ecology, microbiology
community assembly, foliar endophytic fungi,
JanzenConnell hypothesis, Theobroma cacao,
vertical stratification
Author for correspondence:
Natalie Christian
Electronic supplementary material is available
online at
Exposure to the leaf litter microbiome
of healthy adults protects seedlings
from pathogen damage
Natalie Christian1, Edward Allen Herre2, Luis C. Mejia2,3 and Keith Clay1
Evolution, Ecology and Behavior Program, Department of Biology, Indiana University, 1001 East 3rd Street,
Bloomington, IN 47405, USA
Smithsonian Tropical Research Institute, Unit 9100 Box 0948, DPO AA 34002-9998, USA
Institute for Scientific Research and High Technology Services (INDICASAT), Building 219, City of Knowledge,
Clayton, Panama, Republic of Panama
NC, 0000-0003-1568-7645
It is increasingly recognized that microbiota affect host health and physi-
ology. However, it is unclear what factors shape microbiome community
assembly in nature, and how microbiome assembly can be manipulated to
improve host health. All plant leaves host foliar endophytic fungi, which
make up a diverse, environmentally acquired fungal microbiota. Here, we
experimentally manipulated assembly of the cacao tree (Theobroma cacao)
fungal microbiome in nature and tested the effect of assembly outcome on
host health. Using next-generation sequencing, as well as culture-based
methods coupled with Sanger sequencing, we found that manipulating
leaf litter exposure and location within the forest canopy significantly altered
microbiome composition in cacao. Exposing cacao seedlings to leaf litter
from healthy conspecific adults enriched the seedling microbiome with
Colletotrichum tropicale, a fungal endophyte known to enhance pathogen
resistance of cacao seedlings by upregulating host defensive pathways. As
a result, seedlings exposed to healthy conspecific litter experienced reduced
pathogen damage. Our results link processes that affect the assemblyand com-
position of microbiome communities to their functional consequences for host
success, and have broad implications for understanding plant–microbe inter-
actions. Deliberate manipulation of the plant– fungal microbiome also has
potentially important applications for cacao production and other agricultural
systems in general.
1. Background
Exploring the incredible diversity and effects of host-associated microorganisms
is a cornerstone of modern biology. We now know that macroorganisms are
colonized by diverse microbial communities that play an integral role in their
host’s biology. These discoveries have been catalysed by recent advances in
sequencing technologies, which, coupled with traditional culture-based
approaches, are now allowing us to move beyond simple description of the
microbiome to understand its role in host health and physiology. Plant
leaves, for example, are colonized by a remarkably diverse fungal microbiome.
These cryptic symbionts, known as foliar endophytic fungi (FEF), share strong
similarities with other microbiota, such as bacteria, in their ecological organiz-
ation and functional importance [1]. FEF communities can exhibit complex
dynamics, including host specificity [2], priority effects [3], and temporal and
spatial variability [4], and component FEF species can fulfil various symbiotic
functions for their host, including increasing plant vigour [5], drought resist-
ance [6], and enemy defence [710]. A growing body of literature suggests
that many FEF are modifiers of host plant disease severity [11], and multiple
mechanisms of disease suppression by certain endophyte species have been
identified, including by secreting antimicrobial substances [12], out-competing
&2017 The Author(s) Published by the Royal Society. All rights reserved.
on July 8, 2017 from
pathogens [13], or increasing expression of host defences [14].
Although artificial inoculation studies have revealed the
functional roles of some component members of the micro-
biome, the connections between natural FEF community
assembly and their effects on host health have not been
investigated previously.
Like other diverse microbiota, FEF are primarily horizon-
tally transmitted by spores that land on compatible leaf
surfaces, germinate, and penetrate to form localized infec-
tions [1]. This transmission mode allows the fungal
microbiome to be easily manipulated. For instance, FEF-free
plants can be grown in growth chambers and greenhouses
[14] and inoculated with particular fungal species [7,15].
FEF-free seedlings can also be transplanted into the field to
examine how FEF communities form in nature. For instance,
a previous study placed FEF-free cacao tree (Theobroma cacao)
seedlings in a forest where the presence and abundance of
leaf litter within approximately 20 m of the seedlings had
been manipulated [16]. Exposure to leaf litter increased FEF
colonization of tree seedlings, suggesting that FEF recolonize
living tissues of the host or its neighbours from nearby
senesced leaves [16,17]. If local leaf litter is an important
source of FEF colonization, litter identity (i.e. the host species
and its associated microbiota) is also likely to affect FEF com-
munity structure. Moreover, while local leaf litter may be an
important source of FEF near the forest floor, vertical changes
in abiotic conditions potentially filter which microbes suc-
cessfully colonize host tissues at different microsites within
the canopy [17,18], as fungal spore densities are influenced
by abiotic conditions such as humidity, temperature, wind,
light, and canopy drip [19]. However, even if local environ-
mental factors alter FEF community composition, it is
unclear whether such changes affect host health or if differen-
tial outcomes of community assembly are functionally
Here, we investigated whether variation in local leaf
litter and canopy microsite altered the outcome of micro-
biome assembly in cacao (T. cacao). Specifically, we used
endophyte-free T. cacao seedlings as sentinels for FEF coloni-
zation to assess differences in FEF community assembly. We
then tested whether changes in microbiome composition
affected host resistance to the pathogenic oomycete, Phy-
tophthora palmivora, which can infect all cacao tissues and
causes black pod, cacao’s most economically important and
widespread disease [20]. We found that manipulating leaf
litter exposure and location within the forest canopy signifi-
cantly altered microbiome composition in cacao, and that
seedlings exposed to the leaf litter of healthy adults exhibited
reduced pathogen damage. These results are similar to results
from other microbiome systems [21] and suggest that there are
opposing forces (e.g. pathogens and mutualists) acting syn-
chronously within the predictions of the Janzen– Connell
framework for distance-dependent seedling performance in
tropical forests [22].
2. Material and methods
(a) Generation of endophyte-free seedlings
Endophyte-free T. cacao seedlings were generated at the Smith-
sonian Tropical Research Institute in Gamboa, Panama, as
previously described [7,14]. Seeds were collected from T. cacao
trees accession UF12, which were grown in a plantation in
Charagre, Bocas del Toro province, Panama. Cacao seeds were
surface sterilized by submerging them in 0.5% sodium hypo-
chlorite for 3 min, rinsed with sterile water and then placed in
plastic trays of sterilized soil (2 : 1 mixture of clay-rich soil from
Barro Colorado Island, Panama, and rinsed river sand), and ger-
minated in growth chambers. After one month, seedlings were
transplanted into individual 600 ml pots containing the same
soil mixture and returned to growth chambers. Both seed germi-
nation and seedling growth took place in Percival growth
chambers (model I35LL, 115 volts, 1/4Hp, series: 8503122.16,
Percival Scientific, Inc., Perry, IA) with a 12 L : 12 D photoperiod
and temperatures of 308C and 268C, respectively. Germination
in growth chambers has been shown to prevent endophytic
colonization of plant tissue [14]. Prior to experimental manipu-
lation, leaves were tested for FEF colonization. Only 2% of the
leaf fragments tested positively for FEF colonization,
confirming that seedlings were essentially endophyte-free,
consistent with previous studies. Theobroma cacao hosts no
known seed-transmitted fungi.
(b) Experimental treatments and field placement
Potted seedlings were transported in sterile plastic containers to
Barro Colorado Island, Panama, for experimental manipulation.
Seedlings (n¼54) received one of three litter treatments: mixed
species litter (n¼18), T. cacao litter (n¼18), or no litter as a con-
trol (n¼18). Mixed litter was collected from a previously
established long-term ‘litter addition’ experiment on the nearby
peninsula of Gigante. These plots receive bulk compilations of
litter from other experimental plots on the peninsula, and are
relatively well mixed and contain no T. cacao plant material.
Theobroma cacao litter was collected from a healthy, disease-free,
isolated T. cacao tree in Gamboa, Panama. As a result of high neo-
tropical pathogen pressure, we only had access to one healthy
cacao tree growing in nature, as opposed to a highly managed
plantation. ‘No litter’ plants did not receive a litter treatment.
All pots were covered with clean plastic mesh screening at the
base of the plant stem in order to secure the litter treatment (if
present) and to exclude foreign litter and other debris from
the pot.
In May 2014, seedlings were placed in the secondary forest of
Barro Colorado Island, Panama, at three heights within the
canopy: 0 m (ground level), 2 m (low understorey), and 30 m
(upper canopy). Six plants from each litter treatment were
placed at each height, representing a full factorial experimental
design. Vertical stratification was achieved by securing pots to
Lutz Tower, a 48 m canopy tower that sits on a concrete base
within the forest. All litter and debris present at each of the
three heights was cleared away before seedling placement, and
cleared daily during the duration of the experiment. Plants
remained in the field for two weeks and were watered daily at
soil level to avoid artificially wetting the foliage. It did rain reg-
ularly over the course of the experiment, typical of lowland moist
tropical forests in the rainy season. After two weeks, seedlings
were collected and placed in a covered greenhouse for a
pathogen challenge experiment and endophyte community
(c) Pathogen challenge
To test whether litter exposure and canopy microsite affected
host pathogen resistance, a subset of leaves from experimental
plants and FEF-free control plants (n¼40 leaves distributed
across 30 seedlings, including nine FEF-free control leaves dis-
tributed across seven control seedlings) were inoculated with a
strain of P. palmivora previously isolated from symptomatic
T. cacao in Panama. To control for possible effects of leaf age
and development, we only experimentally infected leaves at cer-
tain stages (specifically stages B, C, or D [23]), which are Proc. R. Soc. B 284: 20170641
on July 8, 2017 from
intermediate stages of development, and have been shown to be
most receptive to experimental infection by P. palmivora [23]. To
infect leaves, agar plugs of P. palmivora with mycelia from the
margin of the culture were placed on leaves, which were pricked
with a sterile needle and received an application of 10 ml of sterile
water to facilitate infection. After inoculation, plants were
enclosed in a plastic chamber with wet paper towels to create a
humid environment promoting colonization. After 21 days,
pathogen damage was measured by photographing leaves (elec-
tronic supplementary material, figure S1) and then measuring
the damaged area using IMAGEJ.
(d) Endophyte isolation
To determine whether FEF community composition explained
differences in pathogen damage, one mature leaf per seedling
was harvested for microbiome characterization following the
two-week field exposure. A fully expanded leaf was removed
from each seedling, rinsed under tap water, and processed
within 12 h of collection. Thirty-two 4 mm
tissue fragments
were obtained haphazardly from each leaf and surface sterilized
as follows: tissue fragments were submerged in 70% ethanol for
3 min, 0.525% sodium hypochlorite for 2 min, and then sterile
water for 1 min. Sixteen tissue fragments were stored at 2208C
until used for culture-independent analysis of fungal commu-
nities. The other 16 pieces of tissue were immediately placed in
a grid in 10 cm diameter Petri plates containing 2% malt extract
agar. Plates were sealed with Parafilm and incubated in a growth
chamber with 12 L : 12 D cycle at a constant temperature of 228C.
Plates were monitored daily for fungal growth. Emergent hyphae
were subcultured to new plates and allowed to grow until the
colony covered the agar plate. Vouchers of living mycelia were
suspended in sterile water and stored in the laboratory building
at Barro Colorado Island.
(e) Molecular identification: culture-independent
Total genomic DNA was extracted directly from sterilized plant
tissue using a QIAGEN DNeasy Plant Mini Kit. Primers NSA3
and NLC2 were used to amplify an approximately 1 000 bp
region surrounding the entire internal transcribed spacer (ITS)
region of fungal DNA [24]. Each 25 ml PCR reaction included:
13.38 ml Milli-Q water, 5 ml 5X Green GoTaq
Reaction Buffer,
3ml MgCl
(25 mM), 0.5 ml dNTPs (0.2 mM each dNTP), 1 ml
each primer (5 mM), 0.125 ml GoTaq
DNA Polymerase, and
1ml template. The amplification was run in an MJ Research
Tetrad PTC-225 Thermal Cycler (2.5 min at 958C, followed by
25 cycles (30 s at 958C, 30 s at 60.28C, 45 s at 728C), 5 min at
728C). PCR products were cleaned using an Omega Bio-Tek
Cycle-Pure Kit, and sent to the Biosciences Division
(BIO) Environmental Sample Preparation and Sequencing Facil-
ity (ESPSF) at Argonne National Laboratory for sequencing on
the Illumina MiSeq platform. At Argonne National Laboratory,
genomic DNA was amplified using modified versions of primers
ITS1F and ITS2 [25]. The reverse amplification primer also con-
tained a 12 base barcode sequence that supports pooling of up
to 2 167 different samples in each lane [26,27]. Each 25 ml PCR
reaction consisted of 9.5 ml of MO BIO PCR Water (Certified
DNA-Free), 12.5 ml of QuantaBio AccuStart II PCR ToughMix
(2concentration, 1final), 1 ml Golay barcode tagged Forward
Primer (5 mM concentration, 200 pM final), 1 ml Reverse Primer
(5 mM concentration, 200 pM final), and 1 ml of template DNA.
Amplification was performed as follows: 3 min at 948C, followed
by 35 cycles (45 s at 948C, 60 s at 508C, 90 s at 728C), 10 min at
728C. Amplicons were quantified using PicoGreen (Invitrogen)
and a plate reader. Once quantified, products were pooled into
a single tube at equal concentration. The pool was cleaned up
using AMPure XP Beads (Beckman Coulter), and then quantified
using a fluorometer (Qubit, Invitrogen). After quantification, the
molarity of the pool was determined and diluted to 2 nM,
denatured, and then diluted to a final concentration of 6.75 pM
with a 10% PhiX spike for paired 251-nucleotide read sequencing
on the Illumina MiSeq platform. Reads were demultiplexed, chi-
maeras were removed, and reads were clustered at 97% sequence
identity. Three samples had a very low number of reads and
were removed prior to clustering. Identification of consensus
sequences was performed using the Ribosomal Database Project
(RDP) Bayesian Classifier with the Warcup ITS training set [28],
and archived at GenBank under accession numbers MF148556
(f) Molecular identification: culture-dependent
Total genomic DNA was extracted directly from fungal mycelia
of each fungal isolate using a QIAGEN DNeasy Plant Mini Kit.
Primers ITS5 and ITS4 were used to amplify the ITS region of
fungal DNA. PCR amplifications were achieved using a
Thermo Scientific Phire Plant Direct PCR Kit. Each 20 ml PCR
reaction included: 7.1 ml Milli-Q water, 10 ml2Phire Plant
PCR Buffer (which included dNTPs and MgCl
), 1 mleach
primer (5 mM), 0.4 ml Phire Hot Start II DNA Polymerase, and
0.5 ml template DNA. The amplification was run in an MJ
Research Tetrad PTC-225 Thermal Cycler (30 s at 988C, followed
by 30 cycles (5 s at 988C, 5 s at 628C, 20 s at 728C), 1 min at 728C).
Gel electrophoresis using SYBR Safe produced single bands for
all products, and no bands in negative controls. PCR products
were cleaned using an Omega Bio-Tek MicroElute
Kit and Sanger sequenced for both forward and reverse reads
(primers ITS5 and ITS4, respectively) on an ABI3730 at the
Indiana Molecular Biology Institute.
Of the 335 isolates, 313 high-quality ITS sequences were
obtained. CodonCode Aligner v. 5.0.1 (CodonCode Aligner
Company) was used to make base calls, perform quality assess-
ments, and assemble consensus sequences according to 97% ITS
sequence similarity, with a minimum of 40% overlap [2]. Identi-
fication of consensus sequences was performed using the RDP
Bayesian Classifier with the Warcup ITS training set [28], and
archived at GenBank under accession numbers MF148497
(g) Statistical analyses
The area of pathogen damage (cm
) experienced by each treat-
ment group was compared with baseline pathogen damage in
endophyte-free plants using ANOVA, with litter treatment,
height treatment, and leaf stage as fixed effects. Height was not
significant, nor was there a significant litter height interaction,
so height was removed from the model. Analysis of pathogen
damage was performed using R v. 3.1.2 [29].
For both the culture-independent and culture-dependent
datasets, operational taxonomic units (OTUs), designated by
the ITS region, were used for ecological analyses. Species
accumulation curves and estimates of total richness were inferred
using EstimateS 9.0.1 [30]. Rarefaction curves were scaled by the
number of accumulated samples (i.e. number of host plants
sampled) to depict species density for culture-independent
(electronic supplementary material, figure S2a) and culture-
dependent (electronic supplementary material, figure S2b)
methods [31].
All other community analyses were performed using R
v. 3.1.2 [29]. ANOVA was used to compare culture-based
isolation frequency across height and litter treatments. Assump-
tions of normality and homogeneity of variance were tested and
met. To examine the effects of experimental treatments on FEF Proc. R. Soc. B 284: 20170641
on July 8, 2017 from
community composition, permutational multivariate analysis
of variance (PERMANOVA), using distance matrices was
used with the Bray– Curtis dissimilarity index (VEGAN
Package, function adonis). For the culture-dependent dataset,
PERMANOVA was performed on all non-singleton OTUs, and
for the culture-independent dataset, data were Hellinger-
transformed prior to ordination and diversity analyses. The
ordination goodness-of-fit was measured by the stress value
[32]. Non-metric multidimensional scaling (NMDS) ordinations
were created in VEGAN to visualize community similarities
across height and litter treatments [33]. The Shannon diversity
index was calculated for both the culture-dependent and cul-
ture-independent datasets using the VEGAN Package. Patterns
of co-occurrence of endophytes were analysed within individual
plants in both datasets using C-score analysis, which indicates if
species co-occur more or less often than predicted by a null
model (VEGAN Package, function oecosimu). A structured
community (i.e. significant C-score statistic) implies ecological
mechanisms of community assembly as opposed to purely
random processes. Indicator species analysis was performed on
the culture-independent dataset (INDICSPECIES Package, func-
tion multipatt). For the culture-independent dataset, the number
of reads of the dominant OTU (best hit: Colletotrichum tropicale)
was compared across litter treatments using ANOVA. Height
and total number of reads were included as fixed effects. The
model was also run combining read counts for all OTUs that
had a best match of C. tropicale, but this did not affect the signifi-
cance of the model. Thus, we report results from the model that
included read counts for only the dominant OTU as the response
variable, in order to maintain a conservative estimate of how this
one OTU affected disease outcomes. For the culture-dependent
dataset, the percentage of isolates of the dominant OTU (best
hit: C. tropicale) was compared across litter treatments using
ANOVA. Height treatment was included as a fixed effect.
Additionally, for the culture-independent dataset, a regression
analysis was performed on log-transformed data to test if the
number of reads of the dominant OTU correlated with pathogen
damage across treatments with plant individual as a random
effect. The R
value for the mixed effects model was calculated
using the MuMIn Package (function r.squaredGLMM).
3. Results and discussion
Three weeks after infection by P. palmivora, we measured leaf
necrosis (electronic supplementary material, figure S1), which
differed significantly among litter treatments (d.f. ¼3, F¼
3.133, p¼0.038; figure 1). Plants exposed to cacao litter
experienced significantly less pathogen damage than
FEF-free controls ( p¼0.027). By contrast, damage was not
significantly reduced in the no litter, ( p¼0.947) and mixed
litter ( p¼0.071) treatments. Further, exposure to conspecific
litter reduced pathogen damage to approximately 50% of the
damage on seedlings exposed to mixed litter (figure 1). The
ANOVA included leaf development stage as a fixed effect
(see Material and methods), which was significant (d.f. ¼2,
F¼6.720, p¼0.003). Phytophthora palmivora has a wide
range of dispersal mechanisms, and can be transmitted
through rain, soil, or insects to infect pod, leaf, or seedling
tissues [20]. While it is feasible that pathogen pressure from
P. palmivora may vary throughout the canopy, canopy micro-
site did not significantly affect plant response to pathogen
damage (d.f. ¼2, F¼2.101, p¼0.139) and was removed
from the full model.
To determine whether FEF community composition
explained differences in pathogen damage, we used next-
generation sequencing (NGS) on the Illumina platform, as
well as a culture-based approach coupled with Sanger
sequencing. FEF provide an excellent opportunity to compare
culture-independent and culture-dependent methods, allow-
ing for a more detailed perspective of the plant microbiome.
Most FEF are culturable, so culture-based approaches can
inform abundance and proportion of different FEF species in
tissue, while culture-independent approaches also improve
sequencing depth [1]. Both of these methods are subject to
inherent biases: in culture based-approaches, stronger compe-
titors may emerge from tissue first, to the exclusion of weaker
competitors. In culture-independent approaches, PCR bias
may skew the representation of certain OTUs in the species
pool. Given that we used nested PCR, the high number of
amplification rounds is a potential source of bias. Using
both culture-based and culture-independent methods helps
counterbalance biases inherent to each individual method.
Following culturing, we obtained 335 isolates represent-
ing 59 OTUs (based on Sanger sequencing) from 864 tissue
fragments, with at least one isolate recovered from 51 of 54
leaves (electronic supplementary material, table S1). Iso-
lation frequency differed significantly among litter
treatments (d.f. ¼2, F¼3.841, p¼0.028), with more isolates
obtained from no litter plants compared with plants
exposed to cacao litter ( p¼0.025) (figure 2a). Isolation fre-
quency also varied among microsites (d.f. ¼2, F¼6.164,
p¼0.004), with fewer total isolates obtained from seed-
lings placed high in the canopy compared with low in
the canopy ( p¼0.036) or at ground level ( p¼0.004;
figure 2b). The most abundant OTU in the culture-based
dataset comprised 35% of isolates (116/335), and its best
taxonomic match was C. tropicale.Colletotrichum tropicale is
the dominant species of FEF found in healthy T. cacao
leaves in Panama, and has been previously reported to
enhance pathogen resistance when inoculated as a pure
culture into cacao hosts [15,23].
Independently, using NGS, we obtained 2 127 572 reads
from the other 864 tissue fragments. This method identified
five times more OTUs (294) than the culture-based method
using the same amount of leaf tissue (electronic supplemen-
tary material, table S2). NGS was particularly useful for
identifying rare members of the microbiome, and revealed a
greater total number of OTUs. There was substantial
endophyte-free no litter mixed litter cacao litter
pathogen damage (cm2)
Figure 1. Exposure to cacao litter reduced pathogen damage. Compared with
endophyte-free controls, plants exposed to cacao litter experienced signifi-
cantly less pathogen damage, plants treated with mixed litter experienced
marginally significantly less damage, and no litter plants did not experience
reduced damage. Canopy microsite was not significant. Error bars represent
standard error of the mean. Proc. R. Soc. B 284: 20170641
on July 8, 2017 from
taxonomic overlap between the datasets, with only 5% of iso-
lates identified by Sanger sequencing unrepresented at the
genus level in the NGS approach. Consistent with culture-
based identification, the most abundant OTU produced by
NGS again corresponded to C. tropicale (22% of reads).
Statistical analyses of FEF communities from both the
NGS and culture-based approaches were qualitatively
similar. Litter treatment, microsite, and their interaction sig-
nificantly predicted FEF community composition in the
NGS dataset (PERMANOVA; Litter: F
¼1.888, R
0.063, p¼0.003; Height: F
¼4.359, R
¼0.146, p¼0.001;
Interaction: F
¼1.313, R
¼0.088, p¼0.033; figure 3a–c).
Differences among microsites were qualitatively similar for
the culture-based dataset for non-singleton OTUs (PERMA-
NOVA; Height: F
¼2.715, R
¼0.106, p¼0.003),
although the litter treatment (F
¼0.982, R
¼0.038, p¼
0.447) and the height litter interaction (F
¼1.007, R
0.078, p¼0.457) were not significant in the culture-based
dataset. FEF co-occurred non-randomly compared with a
null model (NGS: C-score ¼17.67, z¼2161.87, p,0.001;
Culture-based: C-score ¼5.71, z¼234.06, p,0.001),
suggesting that treatments affected FEF community assem-
bly. Shannon diversity also differed significantly among
litter treatments (NGS: d.f. ¼2, F¼6.668, p¼0.003; cul-
ture-based: d.f. ¼2, F¼6.231, p¼0.004; figure 2c) and
microsites (NGS: d.f. ¼2, F¼21.342, p,0.001); not signifi-
cant for the culture-based dataset (d.f. ¼2, F¼1.704, p¼
0.192; figure 2d). Specifically, in the NGS dataset, plants in
the no litter treatment harboured more diverse FEF
communities than mixed ( p¼0.019) and cacao ( p¼0.007)
litter treatments, and plants in the canopy had less diverse
FEF communities than those in the understorey ( p¼0.002)
or at ground level ( p,0.001). There was a significant inter-
action between litter and microsite that affected FEF
diversity in the culture-independent dataset (d.f. ¼4, F¼
5.013, p¼0.002). Specifically, FEF communities of no litter
plants maintained high levels of diversity at 30 m, whereas
plants exposed to cacao or mixed litter experienced a signifi-
cant drop in FEF diversity at this height (electronic
supplementary material, figure S3). This interaction was
Shannon diversity
cacao mixed none
ground low high
no. isolates
Figure 2. Litter and microsite affected density and diversity of endophytic
fungi. (a) The number of isolated endophytes differed among litter treat-
ments, with more isolates from no litter plants compared with plants
exposed to cacao litter. (b) The number of isolated endophytes differed
among microsites, with fewer isolates from seedlings placed high in the
canopy compared with low in the canopy or at ground level. (c) Shannon
diversity differed among litter treatments (pictured: NGS dataset). Endophyte
diversity in no litter plants was significantly higher than plants exposed to
cacao or mixed litter. (d) Shannon diversity differed significantly based on
placement in the canopy (pictured: NGS dataset). Endophyte diversity was
lower for seedlings placed high in the canopy compared with low in the
canopy or at ground level. Dots represent individual hosts. Error bars represent
standard error of the mean.
−2 −1 0 1
−2 −1 0 1
−2 −1 0 1
Figure 3. Biotic and abiotic factors affected endophyte community compo-
sition. (a) Litter exposure, (b) canopy microsite, and (c) their interaction
predicted endophyte community composition. NMDS plots are based on
the NGS dataset, but differences were qualitatively similar for the culture-
based dataset for non-singleton OTUs. Error bars represent standard error
of centroids. Proc. R. Soc. B 284: 20170641
on July 8, 2017 from
not significant for the culture-dependent dataset (d.f. ¼4,
F¼0.394, p¼0.812).
Few studies have analysed vertical distribution of FEF in
forests [34], but our results are consistent with a recent study
reporting lower FEF abundance and richness in the canopy of
European Ash [17]. The negative relationship between height
and FEF density and diversity observed here is likely caused
by hotter, drier, and brighter canopy conditions that con-
strain which FEF colonize and grow, despite the presence
of litter in some treatments [19]. While previous work inves-
tigating vertical stratification of FEF communities has focused
on surveying established FEF communities in adult canopy
trees [35], it is not feasible to experimentally manipulate the
endophyte status of individual leaves on adult trees. Instead,
we placed FEF-free seedlings at varying heights in the canopy
as sentinels for FEF colonization. This approach allowed us to
standardize leaf material and control for potential physiologi-
cal differences that could influence de novo endophyte
colonization and pathogen resistance, while also testing the
effect of abiotic conditions on the assembly of FEF commu-
nities. Moreover, P. palmivora infects cacao pods and leaves
throughout a tree [36], so controlling for leaf age and develop-
ment allowed us to test the hypothesis that microbiomes that
assemble at different heights may be more or less effective at
defending plants from pathogens. Our results suggest that
position within the canopy had no effect on seedling pathogen
damage, despite large changes in microbiome composition.
However, the effect of height may have been obscured in
litter-treated plants given that litter was added to pots.
It was unexpected that plants not exposed to litter were
colonized by more abundant and diverse FEF than litter-
treated seedlings, as this contrasts with previous studies
[16], but may reflect differences in the spatial scale of litter
manipulation. While previous work compared FEF coloniza-
tion rates following litter manipulation across large forest
plots, we manipulated litter at the extremely local scale of a
single pot. Our results suggest that when local leaf litter is
present, FEF from the litter quickly colonize nearby host
plants and exert an inhibitory priority effect on later coloni-
zers. Conversely, without local litter inocula as a source of
FEF common to healthy adults, seedlings were colonized
by a greater density and diversity of weedy, highly dispersi-
ble species. This was supported by indicator species analysis,
which revealed more unique taxa associated with no litter
seedlings and seedlings at 30 m (electronic supplementary
material, table S3). Despite strong effects of vertical stratifica-
tion on FEF, differences in pathogen damage were only
attributable to litter treatment. Exposure to litter could have
affected host pathogen response in several ways, including
changing soil nutrient content or leaf chemistry. However,
we found that exposure to conspecific litter significantly
changed the relative abundance of component FEF species,
which significantly correlated with host pathogen resistance.
Colletotrichum tropicale was the most common OTU in
both of our datasets, and has previously been identified as
the most common fungal component of the healthy T. cacao
microbiome in Panama [37]. FEF communities in seedlings
exposed to cacao litter were characterized by increased dom-
inance of C. tropicale (measured in the NGS dataset by the
number of reads of OTU1; d.f. ¼2, F¼3.674, p¼0.034;
figure 4a), despite lower overall FEF density and diversity
(figure 2a,c). Microsite and total number of reads were
included in the ANOVA as fixed effects, and were both
significant (Height: d.f. ¼2, F¼11.405, p,0.001; Total
Number of Reads: d.f. ¼1, F¼4.235, p¼0.046). For the
culture-based dataset, this trend was qualitatively the same.
Moreover, the number of reads of C. tropicale was negatively
correlated with pathogen damage across all treatments (d.f. ¼1,
¼3.740, R
¼0.11, p¼0.053; figure 4b). Taken together,
these results suggest that C. tropicale was acting as a pathogen
inhibitor, and that this effect was the largest in seedlings
exposed to cacao litter, where C. tropicale abundance was
the highest.
We did not experimentally test the specific role of C. tropicale
or other taxa as part of these experiments. However, previous
work has repeatedly shown that artificial inoculations of
C. tropicale reduced severity of P. palmivora damage in leaf
[7] and fruit [15] tissues in cacao, due to upregulation of defen-
sive pathways in cacao [14]. While infection by P. palmivora
triggers an innate plant immune response by upregulating
pathogenesis-related proteins [38], inoculation by C. tropicale
enhances that immune response by inducing the upregulation
cacao mixed
6 000
8 000
10 000
12 000
14 000
Colletotri chum tropicale abundance
(number of OTU1 reads)
culture-independent culture-dependent
−5 0 5 10
OTU1 reads (lo
pathogen damage (cm2)
p= 0.053
R2= 0.11
e of OTU1 isolated
Figure 4. The abundance of C. tropicale correlates with both litter treatment
and pathogen damage. (a) Seedlings exposed to cacao litter were more
strongly dominated by C. tropicale. There were significant differences in
the number of reads of OTU1 (best match C. tropicale) across litter treatments
(plotted in black: number of reads in the NGS dataset). For the culture-based
dataset, this trend was qualitatively the same ( plotted in grey: percentage of
isolates identified as OTU1, for leaves with more than one isolate). Error bars
represent standard error of the mean. (b) Across all treatments in the NGS
dataset, the number of reads of OTU1 (best match C. tropicale) negatively
correlated with pathogen damage. Proc. R. Soc. B 284: 20170641
on July 8, 2017 from
of hundreds of cacao genes, including those related to defence
[14]. Ultimately, this FEF-induced genetic response results in
less severe Phytophthora damage than that of FEF-free plants
[14]. Our results build upon the extensive previous research
on the genetic pathways of T. cacao, and how they are affected
by C. tropicale and P. palmivora, to link multiple levels of bio-
logical organization. Specifically, by identifying how the
component members of the microbiome differentially affect
host gene expression, we may be able to predict the functional
outcomes of microbial community assembly in nature.
While there are other important members of the foliar
microbiome besides fungi, the existing literature has not yet
pointed to a comparable role of these microorganisms in
plant leaves relative to the many well-documented studies
of FEF that demonstrate their important functional roles for
hosts [1]. Based on the results presented here, we suggest
that future studies conduct similar experiments in other
plant systems and with other phyllosphere components,
such as bacteria, viruses, and microbiota with an epiphytic
habit. Additionally, the ecological patterns found in this
study are consistent with other more disparate systems. For
example, our results suggest that litter from healthy con-
specifics triggers priority effects during FEF community
assembly, promoting colonization by species such as
C. tropicale that are effective at inhibiting further microbial
colonization, including infection by pathogens. Priority
effects have been documented for seed-infecting fungal endo-
phytes in some plant species (though not in T. cacao), which
may host one endophyte per seed as a result of strong exclu-
sionary interactions among endophytes [39]. Our results also
directly parallel human microbiome research, which has
demonstrated that vaginally delivered infants are colonized
by a maternal, Lactobacillus-dominated microbiome, while
infants delivered via C-section are more broadly colonized
by the surrounding environment. These differences in coloni-
zation result in greater susceptibility to methicillin-resistant
Staphylococcus aureus infections in C-section newborns [21].
Our results demonstratingincreased pathogen resistance in
seedlings following exposure to healthy conspecific litter
appear to run counter to the predictions of the Janzen– Connell
hypothesis. The Janzen Connell hypothesis proposes that a
tree’s offspring have higher fitness when they are dispersed
farther from the parent, due to localized accumulation of
host-specific enemies [22,40]. Although cacao seedlings near
conspecific adults may experience greater enemy pressure
[41], we found that local FEF communities derived from
healthy conspecific adults can also promote pathogen resist-
ance in conspecific seedlings, potentially by mobilizing plant
defences [14]. Similar patterns are also emerging in below-
ground studies of other plant systems. For instance, the
rhizosphere microbiome can suppress soil-borne pathogens
[42], and recent work showed that arbuscular mycorrhizal
fungi (AMF) can neutralize the Janzen –Connell effect by med-
iating pathogen damage on seedlings [43]. Conversely, another
recent study demonstrated that tree species associated with
AMF have reduced conspecific seedling establishment because
AMF associated with conspecific adults are not as effective at
preventing pathogens compared with ectomycorrhizal hosts
that inhibit pathogen damage to seedlings [44]. Thus, it is
becoming increasingly clear that there are opposing forces
(e.g. pathogens and mutualists) acting synchronously within
the general Janzen–Connell framework. Moreover, while co-
infections by mutualists and pathogens may occur commonly
in nature, such interactions are an underappreciated nuance
of the existing theoretical framework. These considerations
may help to explain why the strength of Janzen –Connell effects
varies across host species or geographical regions [22,41]. Our
results demonstrate that the aboveground plant microbiome
must also be considered when predicting how plant– microbe
interactions affect host health and community interactions
[7,14,16]. Thus, our results have far-reaching implications for
host– microbe interactions, and provide strong impetus for
future ecological research. Our results also suggest that the
application of litter from healthy hosts could be an effective
agricultural strategy to reduce crop losses with smaller
economic and environmental costs than current practices [45].
Data accessibility. Supporting data are available in the electronic sup-
plementary material. Raw sequence data are archived at GenBank
under accession numbers MF148497–MF148849.
Authors’ contributions. N.C. designed and conducted the experiments,
analysed the data, and wrote the first draft. K.C., E.A.H., and
L.C.M. advised on experimental design and provided important
revisions. All authors gave final approval for publication.
Competing interests. We declare we have no competing interests.
Funding. N.C. was supported by an NSF Graduate Research Fellow-
ship. This research was funded by a Smithsonian Tropical Research
Institute Short Term Fellowship (N.C.), and grants from the Myco-
logical Society of America (N.C.), Garden Club of America (N.C.),
and Indiana University (N.C.). This work was also supported by
a grant from the Simons Foundation (429440, WTW).
Acknowledgements. We are grateful to Posy Busby, Luke Henry, and
Briana Whitaker for their feedback on earlier drafts of this manu-
script. Raul Ruiz, Marta Vargas-Timchenko, Enith Rojas, Amanda
Winters, Kristin Saltonstall, Adalberto Gomez, Justin Shaffer, James
DeVore, Courtney Sullivan, and Noelle Visser assisted with the
experiment. Betsy Arnold provided valuable advice. Illumina
sequencing was performed at Argonne National Laboratory, and
the Indiana University Center for Genomics and Bioinformatics
assisted with bioinformatics. Permission to do this research was
granted by the Autoridad Nacional del Ambiente de Panama
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... Experimental evidence demonstrates that fungal endophytes can alter the expression of many genes within the tissues they inhabit (Mejía et al., 2014). Consequently, variation in endophyte communities may influence a range of existing host phenotypic traits (Hawkes et al., 2021), including resistance to herbivory and pathogen attack, (Arnold et al., 2003;Mejía et al., 2008;Telford et al., 2014;Ridout and Newcombe, 2015;Christian et al., 2017), and plant -water relations (Rodriguez et al., 2009;Albrectsen and Witzell, 2012). Given the ecological importance of the foliar endophyte community to the host plant, it is vital that we understand the factors that influence its composition. ...
... Likely explanations for this difference are that additional taxa detected by metabarcoding were either at too low a frequency to be detected in the limited number of culture samples, that the taxa cannot be cultured under the conditions used, or that though they are culturable, they are overgrown and their presence obscured by more vigorous taxa. Other metabarcoding studies concur that hyperdiversity, with many taxa present at very low frequency, is a common feature of fungal endophyte communities that is not revealed by culturing methods Millberg et al., 2015;Christian et al., 2017;Dissanayake et al., 2018;Jayawardena et al., 2018). ...
... A possible consequence for the endophyte community may be an increase in functional redundancy and phylogenetic relatedness because favoured fungal taxa will tend to share common traits that adapt them to the P. sylvestris environment, and/or benefit the host (Shade and Handelsman, 2012). A possible consequence for the host tree is that natural selection could act on ecologically important variation in the endophyte community (Mejía et al., 2014;Christian et al., 2017). Further research is now required to explore these potential ecological and evolutionary consequences of host control of endophyte community composition. ...
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To determine the role of environmental and host genetic factors in shaping fungal endophyte communities we used culturing and metabarcoding techniques to quantify fungal taxa within healthy Scots pine (Pinus sylvestris) needles in a 7-y old provenance-progeny trial replicated at three sites. Both methods revealed a community of ascomycete and basidiomycete taxa dominated by the needle pathogen Lophodermium seditiosum. Differences in fungal endophyte taxon composition and diversity indices were highly significant among trial sites. Within two sites, fungal endophyte communities varied significantly among provenances. Furthermore, the communities differed significantly among maternal families within provenances in 11/15 and 7/15 comparisons involving culture and metabarcoding data respectively. We conclude that both environmental and host genetic variation shape the fungal endophyte community of P. sylvestris needles.
... Culture-dependent methods have been used to identify particular groups of rhizosphere microorganisms, e.g., actinomycetes (Barreto et al. 2008) and arbuscular mycorrhizae (Cuenca and Meneses 1996;Snoeck et al. 2010). Yet amplicon sequencing has been only used to characterize communities associated with other plant and soil regions, such as the phyllosphere (Christian et al. 2017;Santana et al. 2018), endophytic compartments (Rubini et al. 2005;Hanada et al. 2010;Wemheuer et al. 2020), or bulk soil (Mpika et al. 2011;Buyer et al. 2017;Arévalo-Gardini et al. 2020). Numerous studies have also inoculated cacao roots with bacteria and fungi in an attempt to promote plant growth (Supplementary Table 1). ...
... Even with the recent shift from externally applied biocontrol agents to endophytes that may provide longerlasting benefits, Ten Hoopen and Krauss (2016) argue that biological control will only be one component of disease management strategies, as substantial improvements in formulation and application techniques are needed for it to become economically relevant. The native cacao endophyte Colletotrichum tropicale induces systemic changes in gene expression related to pathogen defense (Mejía et al. 2014), and the protection it confers against Phytophthora palmivora can be transferred to endophyte-free seedlings via leaf litter from colonized trees (Christian et al. 2017). Increasing the abundance and persistence in soil of C. tropicale or other rhizosphere microorganisms capable of inducing systemic resistance could therefore be important rhizosphere engineering strategies. ...
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... Plant microbiomes are partitioned by their location above-or belowground, and leaves, in particular, demonstrate high variability (although low within-sample richness) compared with other plant parts (35). This, combined with self-inoculation via litter fall (36), vertical transmission (37), and obligate-coevolved symbioses (38), might all contribute to a significant fraction of the plant microbiome that is unique from other hosts and environments. Although consumers may possess some of these same mechanisms, they appear to contain lower modularity overall. ...
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Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts’ microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.
... Similar findings have recently been reported for Acer saccharum trees [38], where the abundance of conspecific trees in the local metacommunity was positively correlated with the degree of host specialization in the phyllosphere. Moreover, in cacao trees, leaf litter of healthy conspecific hosts was shown to protect against pathogen damage [69]. While we did not test the fitness effects of host filtering for specialized microbial taxa, further investigation of this topic could bring about a new perspective on the conspecific negative density-dependence (i.e., Janzen-Connell) hypothesis, which posits that higher local densities of conspecifics may be disadvantageous due to the possibility of spreading specialized pests or pathogens [29,30]. ...
Microbial communities associated with plant leaf surfaces (i.e., the phyllosphere) are increasingly recognized for their role in plant health. While accumulating evidence suggests a role for host filtering of its microbiota, far less is known about how community composition is shaped by dispersal, including from neighboring plants. We experimentally manipulated the local plant neighborhood within which tomato, pepper, or bean plants were grown in a 3-month field trial. Focal plants were grown in the presence of con- or hetero-specific neighbors (or no neighbors) in a fully factorial combination. At 30-day intervals, focal plants were harvested and replaced with a new age- and species-matched cohort while allowing neighborhood plants to continue growing. Bacterial community profiling revealed that the strength of host filtering effects (i.e., interspecific differences in composition) decreased over time. In contrast, the strength of neighborhood effects increased over time, suggesting dispersal from neighboring plants becomes more important as neighboring plant biomass increases. We next implemented a cross-inoculation study in the greenhouse using inoculum generated from the field plants to directly test host filtering of microbiomes while controlling for directionality and source of dispersal. This experiment further demonstrated that focal host species, the host from which the microbiome came, and in one case the donor hosts’ neighbors, contribute to variation in phyllosphere bacterial composition. Overall, our results suggest that local dispersal is a key factor in phyllosphere assembly, and that demographic factors such as nearby neighbor identity and biomass or age are important determinants of phyllosphere microbiome diversity.
... However, the extent to which fungal endophytes participate in defense reprogramming remains largely unexplored. Most of the existing knowledge supporting the role of fungal endophytes in biotic stress tolerance in forest trees is based on evaluations of external symptoms [31][32][33][34][35], with some but still scarce evidence linking enhanced stress tolerance to the modulation of the plant's defense gene expression caused by the symbiont [36,37]. ...
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Some fungal endophytes of forest trees are recognized as beneficial symbionts against stresses. In previous works, two elm endophytes from the classes Cystobasidiomycetes and Eurotiomycetes promoted host resistance to abiotic stress, and another elm endophyte from Dothideomycetes enhanced host resistance to Dutch elm disease (DED). Here, we hypothesize that the combined effect of these endophytes activate the plant immune and/or antioxidant system, leading to a defense priming and/or increased oxidative protection when exposed to the DED pathogen Ophiostoma novo-ulmi. To test this hypothesis, the short-term defense gene activation and antioxidant response were evaluated in DED-susceptible (MDV1) and DED-resistant (VAD2 and MDV2.3) Ulmus minor genotypes inoculated with O. novo-ulmi, as well as two weeks earlier with a mixture of the above-mentioned endophytes. Endophyte inoculation induced a generalized transient defense activation mediated primarily by salicylic acid (SA). Subsequent pathogen inoculation resulted in a primed defense response of variable intensity among genotypes. Genotypes MDV1 and VAD2 displayed a defense priming driven by SA, jasmonic acid (JA), and ethylene (ET), causing a reduced pathogen spread in MDV1. Meanwhile, the genotype MDV2.3 showed lower defense priming but a stronger and earlier antioxidant response. The defense priming stimulated by elm fungal endophytes broadens our current knowledge of the ecological functions of endophytic fungi in forest trees and opens new prospects for their use in the biocontrol of plant diseases.
Tree planting and natural regeneration contribute to the ongoing effort to restore Earth's forests. Our review addresses how the plant microbiome can enhance the survival of planted and naturally regenerating seedlings and serve in long-term forest carbon capture and the conservation of biodiversity. We focus on fungal leaf endophytes, ubiquitous defensive symbionts that protect against pathogens. We first show that fungal and oomycetous pathogen richness varies greatly for tree species native to the United States ( n = 0–876 known pathogens per US tree species), with nearly half of tree species either without pathogens in these major groups or with unknown pathogens. Endophytes are insurance against the poorly known and changing threat of tree pathogens. Next, we reviewed studies of plant-phyllosphere feedback, but knowledge gaps prevented us from evaluating whether adding conspecific leaf litter to planted seedlings promotes defensive symbiosis, analogous to adding soil to promote positive feedback. Finally, we discuss research priorities for integrating the plant microbiome into efforts to expand Earth's forests. Expected final online publication date for the Annual Review of Phytopathology, Volume 60 is August 2022. Please see for revised estimates.
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Background Scientific approaches into modern agricultural systems, as opposed to the use of synthetic pesticides in food production, became important by exploring endophytic fungi capable of protecting plants against pathogens for maximum crop productivity. Main body Diverse endophytic microbes colonizing the internal tissue of plants exhibit beneficial and pathological effects on plants. The beneficial endophytic fungi assisted plants in the control of pathogenic endophytic fungi in plants due to their ability to directly or indirectly promote plant health. Inefficient agricultural practices and environmental factors contribute to the disease emergence in plants. Endophytic fungi employed diverse mechanisms in phytopathogen control by activating and inducing plant resistance through gene expression, synthesis of fungi-derived metabolite compounds, and hormonal signaling molecules. The mutual coexistence between endophytic fungi and host plants remains an important mechanism in disease suppression. An in-depth understanding and selection of beneficial endophytic fungi and interaction between pathogens and host plants are important in managing challenges associated with the endophyte biocontrol mechanisms. Conclusion Research findings on the use of endophytic fungi as bioinoculants are advancing, and understanding endophytic fungi antibiosis action through the synthesis of biocontrol agents (BCAs) can, however, be explored in integrated plant disease management. Therefore, in this review, the biocontrol mechanism of endophytic fungi against plant pathogens was highlighted.
Manipulating plant microbiomes may provide control of invasive species. Invasive Phragmites australis has spread rapidly in North American wetlands, causing significant declines in native biodiversity. To test microbiome effects on host growth, we inoculated four common fungal endophytes into replicated Phragmites genotypes and monitored their growth in field and growth chamber environments. Inoculations were highly successful in the growth chamber but inoculated plants in the field were rapidly colonized by diverse endophytes from the local environment. There were significant genotype effects and minimal inoculation effects in both experiments with a significant inoculation × genotype interaction on tiller height in the field. Our results demonstrate that endophyte inoculation treatments are feasible, but repeated inoculations may be required to maintain high titer in plants subject to endophyte colonization from the local environment. Future studies should investigate a wider range of fungal endophytes to identify taxa that inhibit Phragmites and other invaders.
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The plant soil feedback (PSF) framework has been instrumental in understanding impacts of soil microbes on plant fitness and species coexistence. PSFs develop when soil microbial communities are altered due to the identity and density of a particular plant species, which can then enhance or inhibit the local survival and growth of that plant species as well as different plant species. The recent extension of the PSF framework to aboveground microbiota, termed here as plant phyllosphere feedbacks (PPFs), can also help determine the impact of aboveground microbes on plant fitness and species interactions. However, experimental tests of PPFs during early plant growth are nascent and the prevalence of PPFs across diverse plant species remains unknown. Additionally, it is unclear whether plant host characteristics, such as functional traits or phylogenetic distance, may help predict the strength and direction of PPFs. To test for the prevalence of litter‐mediated PPFs, recently senesced plant litter from ten native Asteraceae species spanning a range of life history strategies was used to inoculate seedlings of both con‐ and heterospecific species. We found that exposure to conspecific litter significantly reduced growth of four species relative to exposure to heterospecific litter (i.e., significant negative PPFs), three species experienced marginally significant negative PPFs, and the PPF estimates for all ten species were negative. However, neither plant functional traits, nor phylogenetic distance were predictive of litter feedbacks across plant species pairs, suggesting that other mechanisms or traits not measured may be driving conspecific negative PPFs. Our results indicate that negative, litter‐mediated PPFs are common among native Asteraceae species and that they may have substantial impacts on plant growth and plant species interactions, particularly during early plant growth.
Advances in next-generation sequencing have enabled the widespread measurement of microbiome composition across systems and over the course of microbiome assembly. Despite substantial progress in understanding the deterministic drivers of community composition, the role of historical contingency remains poorly understood. The establishment of new species in a community can depend on the order and/or timing of their arrival, a phenomenon known as a priority effect. Here, we review the mechanisms of priority effects and evidence for their importance in microbial communities inhabiting a range of environments, including the mammalian gut, the plant phyllosphere and rhizosphere, soil, freshwaters and oceans. We describe approaches for the direct testing and prediction of priority effects in complex microbial communities and illustrate these with re-analysis of publicly available plant and animal microbiome datasets. Finally, we discuss the shared principles that emerge across study systems, focusing on eco-evolutionary dynamics and the importance of scale. Overall, we argue that predicting when and how current community state impacts the success of newly arriving microbial taxa is crucial for the management of microbiomes to sustain ecological function and host health. We conclude by discussing outstanding conceptual and practical challenges that are faced when measuring priority effects in microbiomes. The order and timing of the arrival (priority effects) of members of a microbiome can influence microbiome composition and function. In this Review, Debray and colleagues provide an overview of the mechanisms of priority effects, highlight examples in host-associated and environmental communities, and discuss methods to detect priority effects in microbial communities.
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Premise of the study: The aboveground tissues of plants host numerous, ecologically important fungi, yet patterns in the spatial distribution of these fungi remain little known. Forest canopies in particular are vast reservoirs of fungal diversity, but intracrown variation in fungal communities has rarely been explored. Knowledge of how fungi are distributed throughout tree crowns will contribute to our understanding of interactions between fungi and their host trees and is a first step toward investigating drivers of community assembly for plant-associated fungi. Here we describe spatial patterns in fungal diversity within crowns of the world's tallest trees, coast redwoods (Sequoia sempervirens). Methods: We took a culture-independent approach, using the Illumina MiSeq platform, to characterize the fungal assemblage at multiple heights within the crown across the geographical range of the coast redwood. Key results: Within each tree surveyed, we uncovered evidence for vertical stratification in the fungal community; different portions of the tree crown harbored different assemblages of fungi. We also report between-tree variation in the fungal community within redwoods. Conclusions: Our results suggest the potential for vertical stratification of fungal communities in the crowns of other tall tree species and should prompt future study of the factors giving rise to this stratification.
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All plants form symbioses with endophytic fungi, which affect host plant health and function. Most endophytic fungi are horizontally transmitted, and consequently, local environment and geographic location greatly influence endophyte community composition. Growing evidence also suggests that identity of the plant host (e.g., species, genotype) can be important in shaping endophyte communities. However, little is known about how disturbances to plants affect their fungal symbiont communities. The goal of this study was to test if disturbances, from both natural and anthropogenic sources, can alter endophyte communities independent of geographic location or plant host identity. Using the plant species white snakeroot (Ageratina altissima; Asteraceae), we conducted two experiments that tested the effect of perturbation on endophyte communities. First, we examined endophyte response to leaf mining insect activity, a natural perturbation, in three replicate populations. Second, for one population, we applied fungicide to plant leaves to test endophyte community response to an anthropogenic perturbation. Using culture-based methods and Sanger sequencing of fungal isolates, we then examined abundance, diversity, and community structure of endophytic fungi in leaves subjected to perturbations by leaf mining and fungicide application. Our results show that plant host individual and geographic location are the major determinants of endophyte community composition even in the face of perturbations. Unexpectedly, we found that leaf mining did not impact endophyte communities in white snakeroot, but fungicide treatment resulted in small but significant changes in endophyte community structure. Together, our results suggest that endophyte communities are highly resistant to biotic and anthropogenic disturbances.
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Background The pathogenesis-related (PR) group of proteins are operationally defined as polypeptides that increase in concentration in plant tissues upon contact with a pathogen. To date, 17 classes of highly divergent proteins have been described that act through multiple mechanisms of pathogen resistance. Characterizing these families in cacao, an economically important tree crop, and comparing the families to those in other species, is an important step in understanding cacao’s immune response. ResultsUsing publically available resources, all members of the 17 recognized pathogenesis-related gene families in the genome of Theobroma cacao were identified and annotated resulting in a set of ~350 members in both published cacao genomes. Approximately 50 % of these genes are organized in tandem arrays scattered throughout the genome. This feature was observed in five additional plant taxa (three dicots and two monocots), suggesting that tandem duplication has played an important role in the evolution of the PR genes in higher plants. Expression profiling captured the dynamics and complexity of PR genes expression at basal levels and after induction by two cacao pathogens (the oomycete, Phytophthora palmivora, and the fungus, Colletotrichum theobromicola), identifying specific genes within families that are more responsive to pathogen challenge. Subsequent qRT-PCR validated the induction of several PR-1, PR-3, PR-4, and PR-10 family members, with greater than 1000 fold induction detected for specific genes. Conclusions We describe candidate genes that are likely to be involved in cacao’s defense against Phytophthora and Colletotrichum infection and could be potentially useful for marker-assisted selection for breeding of disease resistant cacao varieties. The data presented here, along with existing cacao—omics resources, will enable targeted functional genetic screening of defense genes likely to play critical functions in cacao’s defense against its pathogens.
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Many recent studies have demonstrated that non-pathogenic fungi within plant microbiomes, i.e., endophytes ("endo" = within, "phyte" = plant), can significantly modify the expression of host plant disease. The rapid pace of advancement in endophyte ecology warrants a pause to synthesize our understanding of endophyte disease modification and to discuss future research directions. We reviewed recent literature on fungal endophyte disease modification, and here report on several emergent themes: (1) Fungal endophyte effects on plant disease span the full spectrum from pathogen antagonism to pathogen facilitation, with pathogen antagonism most commonly reported. (2) Agricultural plant pathosystems are the focus of research on endophyte disease modification. (3) A taxonomically diverse group of fungal endophytes can influence plant disease severity. And (4) Fungal endophyte effects on plant disease severity are context-dependent. Our review highlights the importance of fungal endophytes for plant disease across a broad range of plant pathosystems, yet simultaneously reveals that complexity within plant microbiomes presents a significant challenge to disentangling the biotic environmental factors affecting plant disease severity. Manipulative studies integrating eco-evolutionary approaches with emerging molecular tools will be poised to elucidate the functional importance of endophytes in natural plant pathosystems that are fundamental to biodiversity and conservation.
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Fungi are key organisms in many ecological processes and communities. Rapid and low cost surveys of the fungal members of a community can be undertaken by isolating and sequencing a taxonomically informative genomic region, such as the ITS (internal transcribed spacer), from DNA extracted from a metagenomic sample, and then classifying these sequences to determine which organisms are present. This paper announces the availability of the Warcup ITS training set and shows how it can be used with the Ribosomal Database Project (RDP) Bayesian Classifier to rapidly and accurately identify fungi using ITS sequences. The classifications can be down to species level and use conventional literature-based mycological nomenclature and taxonomic assignments.
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Plants host a vast diversity of fungal symbionts inside their tissues that live in close proximity with each other to form rich and dynamic communities. Although endophytes can affect plant-herbivore interactions in several ways, it is still not known to what extent such effects are influenced by the properties of endophyte communities or by particular species traits. Here we compared the effects of high versus low foliar fungal endophyte diversity on the preferences of laboratory and wild colonies of leaf-cutting ants. We found that when endophyte densities were high, the ants responded similarly to leaves hosting one endophyte species, Colletotrichum tropicale, or those hosting a species-rich endophyte community. Results were also consistent when comparing the laboratory versus wild ant colonies. We discuss the significance of these results with respect to the ecological effects of plant-endophyte interactions in natural and agricultural ecosystems.
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The field of microbiome research is arguably one of the fastest growing in biology. Bacteria feature prominently in studies on animal health, but fungi appear to be the more prominent functional symbionts for plants. Despite the similarities in the ecological organization and evolutionary importance of animal-bacterial and plant–fungal microbiomes, there is a general failure across disciplines to integrate the advances made in each system. Researchers studying bacterial symbionts in animals benefit from greater access to efficient sequencing pipelines and taxonomic reference databases, perhaps due to high medical and veterinary interest. However, researchers studying plant–fungal symbionts benefit from the relative tractability of fungi under laboratory conditions and ease of cultivation. Thus each system has strengths to offer, but both suffer from the lack of a common conceptual framework. We argue that community ecology best illuminates complex species interactions across space and time. In this synthesis we compare and contrast the animal-bacterial and plant–fungal microbiomes using six core theories in community ecology (i.e., succession, community assembly, metacommunities, multi-trophic interactions, disturbance, restoration). The examples and questions raised are meant to spark discussion amongst biologists and lead to the integration of these two systems, as well as more informative, manipulatory experiments on microbiomes research.
Feedback with soil biota is an important determinant of terrestrial plant diversity. However, the factors regulating plant-soil feedback, which varies from positive to negative among plant species, remain uncertain. In a large-scale study involving 55 species and 550 populations of North American trees, the type of mycorrhizal association explained much of the variation in plant-soil feedbacks. In soil collected beneath conspecifics, arbuscular mycorrhizal trees experienced negative feedback, whereas ectomycorrhizal trees displayed positive feedback. Additionally, arbuscular mycorrhizal trees exhibited strong conspecific inhibition at multiple spatial scales, whereas ectomycorrhizal trees exhibited conspecific facilitation locally and less severe conspecific inhibition regionally. These results suggest that mycorrhizal type, through effects on plant-soil feedbacks, could be an important contributor to population regulation and community structure in temperate forests.
Nonpathogenic foliar fungi (i.e. endophytes and epiphytes) can modify plant disease severity in controlled experiments. However, experiments have not been combined with ecological studies in wild plant pathosystems to determine whether disease-modifying fungi are common enough to be ecologically important. We used culture-based methods and DNA sequencing to characterize the abundance and distribution of foliar fungi of Populus trichocarpa in wild populations across its native range (Pacific Northwest, USA). We conducted complementary, manipulative experiments to test how foliar fungi commonly isolated from those populations influence the severity of Melampsora leaf rust disease. Finally, we examined correlative relationships between the abundance of disease-modifying foliar fungi and disease severity in wild trees. A taxonomically and geographically diverse group of common foliar fungi significantly modified disease severity in experiments, either increasing or decreasing disease severity. Spatial patterns in the abundance of some of these foliar fungi were significantly correlated (in predicted directions) with disease severity in wild trees. Our study reveals that disease modification is an ecological function shared by common foliar fungal symbionts of P. trichocarpa. This finding raises new questions about plant disease ecology and plant biodiversity, and has applied potential for disease management.