INSIGHTS
Red coloration in young tropical leaves associated with reduced fungal
pathogen damage
Peter Tellez1, Enith Rojas2, and Sunshine Van Bael1,2,3
1 Department of Ecology and Evolutionary Biology, Tulane University, 6823 St. Charles Avenue, New Orleans, LA, 70118-5698, U.S.A.
2 Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Balboa, Ancon, Republic of Panama
ABSTRACT
The adaptive significance of red coloration in tropical forest leaves remains unclear. In vivo assays show that there is a significant negative
correlation between anthocyanin pigments in young leaves and fungal pathogen damage. This supports a previous hypothesis that antho-
cyanins may protect young leaves from fungal damage during the vulnerable period of leaf expansion.
Abstract in Spanish is available with online material.
Key words: anthocyanins; Calonectria sp; canopy crane system; Panama; tropical wet forest.
IN THE HUMID TROPICS, APPROXIMATELY ONE-THIRD OF WOODY
PLANT SPECIES DELAY GREENING OF THEIR LEAVES via delayed pro-
duction of chlorophyll pigments or chloroplast development
(Coley & Kursar 1996), and often exhibit a dramatic red col-
oration in their young leaves (Coley & Barone 1996). This red
coloration is attributed to high concentrations of anthocyanin pig-
ments in mesophyll and epidermal cells produced during leaf
expansion that disappear as the leaf matures (Coley & Barone
1996). Anthocyanin pigments have been well studied in plants for
the role they play in attracting pollinators and frugivores (Lat-
tanzio et al. 2006) but their functional significance in young devel-
oping leaves is not entirely understood.
Several hypotheses have been proposed to explain the adap-
tive significance of anthocyanin pigments in young leaves. Antho-
cyanins may protect leaves against photoinhibition (Gould et al.
1995, Close & McArthur 2002, Close & Beadle 2003) or act as a
photoprotectant for plant organelles against UV radiation (Lee &
Lowry 1980, Burger & Edwards 1996). These two hypotheses
are unlikely in the tropics. Xanthophylls, and not anthocyanins,
are known to protect against photoinhibition in tropical forests
(Krause et al. 1995). In addition, if anthocyanins were photopro-
tective, we would expect to find red leaves more often in the
canopy—where brighter conditions exist—relative to the under-
story, but this has not been the case (Dominy et al. 2002). Alter-
natively, it has been suggested that anthocyanins defend against
herbivory, with their pigments providing cryptic coloration to
insect herbivores that are blind to the red part of the light spec-
trum (Dominy et al. 2002, Karageorgou & Manetas 2006).
Although anthocyanin pigments may act as a deterrent to herbi-
vores, few studies have examined the role of anthocyanins in pro-
tecting plants against pathogens.
In tropical rain forests, fungal pathogens play a major role
in shaping and maintaining plant diversity and species composi-
tion (Bagchi et al. 2014), negatively affecting many plants through
reduced host growth or reproduction (Gilbert 1995, Bagchi et al.
2014, Garc
ıa-Guzm
an & Heil 2014). Plants are known to protect
themselves from pathogens through the production of phenolic
compounds such as flavonoids, which have anti-fungal properties
(Alcerito et al. 2002, Lattanzio et al. 2006, Treutter 2006). Seed-
lings with anthocyanin pigments—a sub-group of flavonoids—
have been associated with higher survival rates compared to seed-
lings with green leaves (Queenborough et al. 2013). This could be
driven in part by anthocyanin mediated protection against patho-
gens. Coley and Aide (1989) found that leaf-cutter ants preferen-
tially picked up leaves without anthocyanins. They further
hypothesized that anthocyanins may protect against fungal patho-
gen attack during the vulnerable period of leaf expansion, but no
follow-up study has been conducted. Our study explores this
hypothesis by performing in vivo bioassays with a generalist fungal
pathogen and young leaves varying in anthocyanin content. In a
wet evergreen forest in Panama, we examine the question, do
anthocyanins in developing young leaves protect against fungal
pathogen damage?
The study was conducted in Parque Nacional San Lorenzo
(PNSL) in the Republic of Panama. Parque Nacional San Lor-
enzo (9°170N, 79°580W) encompasses 5.96 ha of tropical, wet,
evergreen forest on the Caribbean coast of Panama. This site
receives approximately 2700–3000 mm of annual rainfall and has
a mean annual temperature of 26°C with little variation among
months (Condit et al. 2004). The site plot is 400 9 100 m with a
140 9 140 m contiguous area to the left side of the southern-
most hectare. A canopy research crane managed by the Smithso-
nian Tropical Research Institute found in the center of this
140 9 140 m plot gives canopy access for an area of 0.9 ha
(Condit et al. 2004).Received 5 July 2015; revision accepted 14 October 2015.
3Corresponding author; e-mail: svanbael@tulane.edu
150 ª 2016 The Association for Tropical Biology and Conservation
BIOTROPICA 48(2): 150–153 2016 10.1111/btp.12303
To determine the best generalist pathogen to use in our field
inoculations, we tested eight fungal pathogens and control plugs
without fungal pathogens on three leaves from various tree spe-
cies near our lab in Gamboa, Panama. A species of Calonectria
was shown to infect all of the leaves from all trees sampled. Sev-
eral species of Calonectria are known necrotrophic plant pathogens
associated with a wide range of agricultural and forestry crops
worldwide (Lombard et al. 2010), with leaf spotting being the
most common disease symptom. Five tree species in PNSL were
chosen for fungal inoculation trials: Brosimum utile (Moraceae),
Coccoloba sp. (Polygonaceae), Perebea angustifolia (Moraceae), Protium
panamense (Burseraceae), and Manilkara bidentata (Sapotaceae).
These tree species were chosen because they were flushing an
abundance of young leaves, showed variation in leaf color, and
were easy to access using the canopy crane system.
We prepared pathogen inoculum following Gilbert and
Webb (2007). This involved placing caps of 1.8 ml cryovials
(Sigma, St Louis, Missouri, U.S.A.) inside large glass Petri dishes
with the deep end of the cryovial cap facing upward. The cryovial
caps were autoclaved for 20 min and, using a pipette, we trans-
ferred sterile, molten, 2% malt extract agar (MEA) into each cap
until full. Once the agar had solidified, we placed a small piece
(~2–3 mm2) of colony cut from 1-wk old Calonectria sp. cultures
grown on 2% MEA on solidified caps. Petri plates containing
caps with fungi were placed inside an incubator (23°C) and
allowed to grow for 5 d before inoculations. Once fungi had
grown to cover the caps, we removed the caps and placed them
in sterile, sealed Whirl-pak bags (Nasco, Fort Atkinson, Wiscon-
sin, U.S.A.) for transport to the study site.
We randomly selected ten young leaves from five tree spe-
cies (N = 100) from both the forest understory and canopy for
inoculation trials. We surveyed trees for newly flushed leaves by
visually inspecting for leaf color and size (30–50% of full size),
relative to older, mature leaves. Before pathogen inoculation, we
measured anthocyanin content on each leaf at three sites: apically,
medially, and basally, and averaged measurements together. About
80–90 percent of anthocyanins found in leaves are composed of
cyanidin, a type of anthocyanin with a transmittance wavelength
of 520–530 nm (Coley & Aide 1989, Onslow 2014). We mea-
sured anthocyanin content using the ACM-200 plus (Opti-
Sciences Inc., Hudson, New Hampshire, U.S.A.). The ACM-200
uses the ratio of % transmittance at a wavelength of 931 nm (in-
frared wavelength to compensate for sample thickness) and
525 nm (anthocyanin transmittance) to calculate an anthocyanin
content index (AIC) value (AIC = % transmittance at 931 nm/%
transmittance at 525 nm). We used sterile forceps to remove the
inoculum cap from the Whirl-pak, pressed the cap against the
underside of the leaf, and clipped it in place with a bent, snap-on
hair clip (Scunci, East Windsor, New Jersey, U.S.A.). Leaves were
harvested after 4 d and the area of visible necrosis damage was
measured using the software Image J (NIH, U.S.A.). All infected
leaves were removed from the forest and transported to our lab
in Gamboa for measurement and disposal.
A Shapiro–Wilk’s test showed that the continuous variables
of leaf damage and anthocyanin content were not normally dis-
tributed. Transformation did not remedy their non-normality. A
non-parametric Levene’s test verified the equality of variances in
the samples. Non-parametric Spearman’s rank correlation was
performed to examine the relationship between foliar fungal
pathogen damage and anthocyanin content in leaves. In addition,
a Mann–Whitney U-test was used to test for differences in antho-
cyanin content and pathogen damage between the forest canopy
and understory. The data were analyzed in SPSS v. 21.1 for Win-
dows (SPSS, Chicago, Illinois, U.S.A.).
Sixty-two leaves were recovered and all leaves had visible
necrosis damage. The inoculation trials using the fungal pathogen
Calonectria sp. showed a significant negative correlation between
anthocyanin content in young leaves and necrosis damage by the
fungal pathogen (r = 0.459, N = 62, P < 0.001, Fig. 1). The
relationship appears as a negative exponential (Fig. 1), however,
we did not fit a negative exponential line because our data vio-
lated the normality assumption. Overall, an increase in antho-
cyanin content led to a decrease in necrosis damage. Tree species
varied in the amount of anthocyanin content and necrosis dam-
age (Table 1). Among the tree species, Protium panamense had the
highest anthocyanin content in both the forest canopy and under-
story but had middle range values for leaf damage (Table 1).
Young leaves of Coccoloba sp. also had higher levels of antho-
cyanins and showed the lowest necrosis damage in the canopy
(Table 1).
A comparison of young leaves showed that there was no sig-
nificant difference in anthocyanin content between the forest
understory (median = 3.26) and the forest canopy (me-
dian = 4.63), (U = 359.0, P = 0.148). However, when comparing
leaf damage, young leaves in the canopy (median = 2.49) had
Anthocyanin content
0 2 4 6 8 10 12 14 16 18 20
Le
af
 a
re
a 
w
ith
 d
am
ag
e 
(cm
2 )
0
2
4
6
8
10
12
14
FIGURE 1. Relationship between anthocyanin content in young leaves and
pathogen induced damage. Data are shown for 62 leaves of five tropical tree
species from both the forest canopy and understory.
Red Coloration in Tropical Plants 151
significantly less necrosis damage than leaves found in the forest
understory (median = 3.77) (U = 230.0, P = 0.002, r = 0.38).
In this study, we found that leaf necrosis damage by the fun-
gal pathogen Calonectria sp. decreased as anthocyanin content in
young leaves increased, supporting the hypothesis that antho-
cyanin pigments may act as an anti-fungal agent to protect young
developing leaves in the tropics (Coley & Aide 1989). In the trop-
ics, almost 70 percent of a leaf ’s lifetime damage occurs during
the small window of leaf expansion when young leaves lack
developed cuticles and lignified cell walls (Coley & Aide 1989,
Coley & Barone 1996). All plants encounter numerous pathogens
in a natural environment and are known to produce secondary
metabolites such as phenols, tannins, and flavonoids, to limit or
inhibit pathogen attack (Lattanzio et al. 2006). Previous research
has shown that anthocyanins—which are secondary metabolites
belonging to the parent group flavonoids—reduce susceptibility
to fungal pathogens in fruits and vegetative tissues (Schaefer et al.
2008, Hafidh et al. 2011). For example, fruit-rot in grape varieties
infected with the necrotrophic fungus Botrytis cinerea was lower
for grapes with high concentrations of anthocyanin compared to
grapes with low anthocyanin concentrations (Schaefer et al. 2008),
and anthocyanin extracts of red cabbage leaves inhibited fungal
mycelial growth in in vitro bioassays (Hafidh et al. 2011). Red col-
oration by anthocyanin pigments could also provide a similar
defensive function in developing young leaves when the risk of
pathogen attack is high (Garc
ıa-Guzm
an & Dirzo 2001). Our
results, illustrated by Fig. 1, also show a similar pattern found by
Schaefer et al. (2008) in grapes with high anthocyanin pigments,
namely that the relationship between anthocyanin concentrations
and fungal concentration was a negative exponential rather than
linear. This may indicate that high anthocyanin content in young
leaves is sufficient defense against pathogen attack; however, the
broad range of damage in leaves with low anthocyanin content
indicates that leaves use alternative anti-fungal compounds for
defense.
We found significantly lower levels of necrosis damage to
young leaves in the forest canopy relative to the understory.
Canopy leaves are smaller, tougher and have higher phenolic con-
tents relative to understory leaves (Coley & Barone 1996) which
may make it harder for fungal pathogens to enter and extend
throughout the leaf tissue. Moreover, fungal pathogen infection
relies on moisture, favorable temperatures, and high relative
humidity (Coley & Barone 1996), environmental conditions often
lacking in the generally hot and dry forest canopy. Our results
are in contrast to findings by Garc
ıa-Guzm
an and Dirzo (2004)
and Gilbert (1995) which found higher incidences of fungal dis-
ease symptoms in the canopy compared to the forest understory.
One explanation from Gilbert (1995) was that higher incidences
of canopy herbivory create leaf wounds that make canopy leaves
more susceptible to pathogens. Garc
ıa-Guzm
an and Heil (2013)
observed that the pattern of fungal incidence between the forest
understory and canopy changes when separating biotrophic from
necrotrophic fungal pathogens. Necrotrophic pathogens are more
commonly reported to infect shade-tolerant plants in the under-
story (Garc
ıa-Guzm
an & Heil 2013), and our findings with
necrotrophic Calonectria sp. are in line with this general pattern.
Leaves in the canopy also contain high levels of phenolic
compounds (Coley & Barone 1996). Phenolic compounds such
as anthocyanins may be present in higher concentrations in the
forest canopy relative to the understory, but in this study there
was no significant difference in anthocyanin content between
canopy and understory leaves. These results are similar to those
found by Dominy et al. (2002), which found that the canopy had
no greater tendency to flush red than did the understory trees.
We found support for the hypothesis that fungal pathogen
damage is negatively associated with anthocyanin content in
young tropical leaves, but there are a few caveats. First, our study
used a single generalist fungal pathogen, which cannot represent
the wide variety of fungal pathogens in a tropical forest. Second,
we surveyed a relatively small number of tree species in Parque
Nacional San Lorenzo. Third, we did not directly test the toxicity
of anthocyanins on our fungal pathogen. Last, we only measured
anthocyanin pigments having a 525 nm or greater absorption
wavelength. This may not be representative of the diverse group
of anthocyanin pigments found within leaves that contribute to
leaf defense. Further research testing how anthocyanins affect
fungal pathogens must expand to include a wider range of fungal
pathogens, including host-specific species, as well as a greater
selection of tree species. Moreover, to examine whether resistance
to fungi is caused by anthocyanins rather than other leaf proper-
ties, anthocyanins alone could be tested on various fungi grown
in vitro. Despite these limitations, our study expands our under-
TABLE 1. Data for mean (SE) anthocyanin content (AIC) and fungal pathogen damage (cm2, leaf area) for five tree species located in the forest canopy and understory at Parque
Nacional San Lorenzo.
Species
Canopy Understory
N AIC Damage N AIC Damage
Brosimum utile 9 4.10 (0.35) 2.62 (0.31) 5 4.32 (0.55) 2.82 (0.34)
Coccoloba sp. 5 9.85 (0.69) 1.95 (0.18) 5 4.60 (0.37) 3.13 (0.48)
Protium panamense 9 14.8 (1.03) 2.79 (0.18) 3 7.14 (2.75) 4.30 (0.89)
Manilkara bidentata 5 2.55 (0.07) 2.29 (0.43) 7 2.48 (0.68) 5.52 (0.94)
Perebea angustifolia 6 2.52 (0.41) 4.63 (0.56) 5 2.57 (0.19) 8.99 (0.89)
Mean 6.76 (0.51) 2.86 (0.33) 4.22 (0.90) 4.95 (0.74)
152 Tellez, Rojas, and Van Bael
standing of the role that anthocyanins play in influencing plant-
fungal interactions and provides greater insight into the selective
factors for red leaf coloration in the humid tropics.
ACKNOWLEDGMENTS
We thank Joe Wright, Mirna Samaniego, Emma Tower, Kimberly
Mighell, Gloribel Vergara Guerrero, and Elizabeth Kimbrough
for their help and for comments on early drafts. We thank the
Smithsonian Tropical Research Institute (STRI) and the Tropical
Canopy Biology Program at STRI for their support. Funding was
from NSF-DEB-0949602 to SAV, Tulane University (The School
of Science and Engineering and the Stone Center for Latin
American Studies) and STRI.
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