Fungal endophyte diversity in coffee plants from Colombia,
Hawai’i, Mexico and Puerto Rico
Fernando E. VEGAa,*, Ann SIMPKINSa, M. Catherine AIMEb,1, Francisco POSADAc,
Stephen W. PETERSONd, Stephen A. REHNERb, Francisco INFANTEe, Alfredo CASTILLOe,
A. Elizabeth ARNOLDf
aSustainable Perennial Crops Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Bldg. 001, BARC-W, Beltsville,
MD 20705, USA
bSystematic Mycology and Microbiology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Bldg. 011A, BARC-W,
Beltsville, MD 20705, USA
cCentro Nacional de Investigaciones de Cafe ´, Chinchina ´, Caldas, Colombia
dMicrobial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, U. S. Department of Agriculture,
Agricultural Research Service, 1815 N. University St., Peoria, IL 61604, USA
eEl Colegio de la Frontera Sur (ECOSUR), Carretera Antiguo Aeropuerto Km. 2.5, Tapachula, 30700 Chiapas, Me ´xico
fDivision of Plant Pathology and Microbiology, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA
a r t i c l e i n f o
Received 24 February 2009
Revision received 10 July 2009
Accepted 13 July 2009
Available online 1 October 2009
Corresponding editor: Kevin Hyde
a b s t r a c t
Coffee (Coffea arabica) plant tissues were surface-sterilized and fungal endophytes isolated
using standard techniques, followed by DNA extraction and sequencing of the internal
transcribed spacer region (ITS). A total of 843 fungal isolates were recovered and sequenced
(Colombia, 267; Hawai’i, 393; Mexico, 109; Puerto Rico, 74) yielding 257 unique ITS
genotypes (Colombia, 113; Hawai’i, 126; Mexico, 32; Puerto Rico, 40). The most abundant
taxa were Colletotrichum, Fusarium, Penicillium, and Xylariaceae. Overall, 220 genotypes were
detected in only one of the countries sampled; only two genotypes were found in all four
countries. Endophytes were also isolated from Coffea canephora, Coffea congensis, Coffea
liberica, Coffea macrocarpa, Coffea racemosa, and Coffea stenophylla in Hawai’i. The high
biodiversity of fungal endophytes in coffee plants may indicate that most of these are
‘‘accidental tourists’’ with no role in the plant, in contrast to endophytes that could be
defined as ‘‘influential passengers’’ and whose role in the plant has been elucidated. This
study, the most comprehensive analysis of fungal endophytes associated with a single host
species, demonstrates that coffee plants serve as a reservoir for a wide variety of fungal
endophytes that can be isolated from various plant tissues, including the seed, and
illustrates the different fungal communities encountered by C. arabica in different coffee-
growing regions of the world.
ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.
* Corresponding author. Tel.: þ1 301 504 5101; fax: þ1 301 504 1998.
E-mail address: email@example.com (F.E. Vega).
1Present address: Department of Plant Pathology and Crop Physiology, Louisiana State University AgCenter, 302 Life Sciences Bldg.,
Baton Rouge, LA 70803, USA
1754-5048/$ – see front matter ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/funeco
fungal ecology 3 (2010) 122–138
The genus Coffea (Rubiaceae) comprises 103 species from
tropical Africa, Madagascar, and the Mascarene Islands
(Davis et al. 2006). Two species, Coffea arabica and Coffea
canephora (also known as robusta) make coffee the second
largest export commodity in the world after petroleum
products, with an estimated annual retail sales value of US
$70 billion (Lewin et al. 2004). C. arabica is endemic to
Ethiopia, SE Sudan, and northern Kenya, while C. canephora is
endemic to various countries throughout tropical Africa
(Davis et al. 2006). Coffee is planted in more than 10 million
hectares in over 50 countries (http://faostat.fao.org), and
approximately 125 million people in Latin America, Africa,
and Asia are dependent on coffee for their subsistence
(Osorio 2002; Lewin et al. 2004).
While a diversity of fungal pathogens associated with
coffee has been recorded in the literature (e.g., Muller et al.
2004), there is a paucity of information regarding nonpatho-
genic, symbiotic fungi such as endophytes associated with
these economically important plants. Fungal endophytes
have been defined in many ways (Schulz & Boyle 2006; Hyde &
Soytong 2008), however, broadly defined, fungal endophytes
are ‘‘fungi . which for all or part of their life cycle invade
the tissues of living plants and cause unapparent and
asymptomatic infections entirely within plant tissues, but
cause no symptoms of disease’’ (Wilson 1995). Recent studies
have shown that some fungal endophytes can protect host
plants against pathogens and herbivores (e.g., Freeman &
Rodriguez 1993; Arnold et al. 2003; Arnold & Lewis 2005; Schulz
& Boyle 2005) and in some cases include entomopathogenic
species (Arnold & Lewis 2005; Vega et al. 2008b). Neither the
ecological importance nor economic applications of most
endophytes havebeenresolved,however, in partbecausevery
few plant species have been comprehensively surveyed for
endophytic fungi. Given the tremendous economic impor-
tance of coffee, the wide geographic range of the genus, and
the importance of the plant in sustainable agroforestry and
conservation efforts (Wintgens 2004; Perfectoet al. 2007), there
is growing enthusiasm to examine the endophyte communi-
ties associated with Coffea.
Rayner (1948), working in Kenya, published the first paper
on coffee endophytes after surface-sterilizing healthy leaves,
pedicels, stems, and green berries with a mercuric chloride–
saponin solution. That study recovered the causal agent of
coffee berry disease, Colletotrichum coffeanum, (currently
known as Colletotrichum kahawae Waller & Bridge), and species
of Phoma and Phomopsis. Santamarı ´a & Bayman (2005) reported
Botryosphaeria, Colletotrichum, Guignardia, and Xylaria species
as fungal endophytes in coffee plants from Puerto Rico. Vega
et al. (2006) identified 13 Penicillium species as endophytes in C.
arabica, Coffea congensis, Coffea dewevrei and Coffea liberica,
including Penicillium coffeae, a new species described by
Peterson et al. (2005). In a study describing the introduction
of the fungal entomopathogen Beauveria bassiana as an
endophyte in coffee plants, Posada et al. (2007) reported the
presence of more than 35 fungal endophytes from coffee
seedlings purchased at a plant nursery in Maryland; they
hypothesized that the presence of these endophytes might
have prevented B. bassiana from becoming established in the
plants. Vega et al. (2008a) also detected seven genera of fungi
as endophytesingreen coffee
Guatemala, India, Kenya, Papua New Guinea, Puerto Rico, and
Vietnam. Several genera of entomopathogenic fungi in coffee
plants were alsorecovered
a different study (Vega et al. 2008b). Similarly, Vega et al. (2005)
reported the presence of 19 genera of bacterial endophytes in
coffee plants from Colombia, Hawai’i, and Mexico. To date,
however, comprehensive studies assessing the geographic
heterogeneity of Coffea endophytes among different coffee-
growing regions of the world, the similarity of endophyte
communities among different sympatric species of Coffea, and
the tissue specificity of endophytes inhabiting C. arabica have
not been conducted.
Inthis paper,wereportthefirstresults ofasurveyoffungal
endophytes associated with various asymptomatic tissues of
coffee plants in Colombia, Hawai’i, Mexico, and Puerto Rico.
from variouslocations in
Materials and methods
Coffee plants (C. arabica) were sampled for endophytic fungi in
2002 and 2003. Sampling sites included one location in
Colombia (National Coffee Research Center (CENICAFE´),
Chinchina ´, Caldas; Jul. 2003); 10 locations throughout Hawai’i
(Jan. 2003); six locations in Chiapas, Mexico (Sep. 2002); and
one location in Puerto Rico (Jun. 2002; Table 1). To facilitate
discussion, Colombia, Hawai’i, Mexico, and Puerto Rico are
referred to as ‘‘countries’’ throughout the paper. At various
Hawai’i locations, other Coffea species were also sampled:
C. canephora, C. congensis, C. liberica, C. macrocarpa, C. racemosa,
and C. stenophylla (Table 1).
Various asymptomatic parts of coffee plants were sampled,
including leaves, roots, stems, and berries. Berries were
divided into various sections: crown, sections of the berry
itself, seeds, and the peduncle. Mature leaves were collected
from the middle section of each plant at the approximate mid-
point of the branch. Age of plants could not be determined,
and collection sites varied in levels of shading from fully
shaded to full sun. No attempts were made to collect only
under one condition. Not all tissues were sampled in each
Plant parts were washed in running tap water and
sectioned into small pieces under sterile conditions with
a sterile scalpel (see Posada et al. 2007 for standard isolation
methods). Sections were surface-sterilized by dipping in 0.5 %
sodium hypochlorite for 2 min and 70 % ethanol for 2 min
(Arnold et al. 2003), and rinsed in sterile distilled water before
surface-drying on sterile paper. Sections were plated on yeast
malt agar (YMA; Sigma Aldrich Co., St. Louis, MO) to which
0.1 % stock antibiotics was added (see Vega et al. 2005). Plates
were kept at room temperature for several months, and any
fungal growth was subcultured onto individual YMA plates for
subsequent DNA extraction. Because the vast majority of
Fungal endophyte diversity in coffee plants 123
fungi did not sporulate in culture, we characterized all isolates
using molecular sequence data.
DNA extraction, amplification, and sequencing
Endophytes were grown in potato dextrose broth (Difco,
Becton Dickinson, Sparks, MD) at 125 rpm on an Innova 4000
Incubator Shaker (New Brunswick Scientific Co., Inc., Edison,
NJ) at 25?C for one week. Fungal tissue was then harvested,
lyophilized, and stored at ?80?C. Lyophilized tissues were
also intended for re-growth of the isolates and subsequent
deposit in culture collections. Unfortunately, most tissues
were not viable, except for several Penicillium and Aspergillus
species that have been deposited in the NRRL collection
(Table 1). For DNA extraction, ca. 50 mg of lyophilized myce-
lium were placed in a 2 ml microcentrifuge tube with
ca. 0.2 ml 1.0 mm zirconia-glass beads (Cat # 1107911-0z,
BIOSPEC, Bartlesville, OK). The mycelium was crushed with
a plastic pestle and further ground in a Fast-Prep-120 sample
grinder (Q-BIOgene, Irvine, CA) for 3 sec at the maximum
speed setting of 6. The powdered mycelium was suspended in
700 mL detergent solution (2 M NaCl, 0.4 % w/v deoxycholic
acid–sodium salt, 1 % w/v polyoxyethylene 20 cetyl ether) and
then agitated for 14 s in the Fast-Prep at maximum speed.
Vials were incubated for 5 min at 55?C in a heat block and
then centrifuged at 7000 g for 5 min followed by emulsion
with 700 ml chloroform/isopropyl and centrifugation at 7000 g
for 5 min. The aqueous phase was transferred to a clean tube
to which an equal volume of 6 M guanidinium thiocyanate
was added. Fifteen microliters of silica powder were gently
mixed with the solution while incubating at room tempera-
ture for 5 min, followed by 3 s centrifugation, after which the
supernatant was discarded. The glass powder was rinsed
twice by suspending in 750 mL ethanol buffer (10 mM Tris–HCl,
pH 8.0, 0.1 mM EDTA, 50 % ethanol) with a disposable transfer
pipette,then collected by centrifugation. The supernatant was
discarded, and the glass powder pellet was dried on a heat
block at 55?C for 5–10 min. The glass powder was re-hydrated
with 105 mL ultra-pure water and genomic DNA eluted by
incubating on a heat block at 55?C for 5–10 min. Following
vortexing and centrifugation, 100 mL of the aqueous superna-
tant was transferred to a clean tube. In a few instances, DNA
extractions were made directly from fungal cultures grown on
potato dextrose agar (Difco, Becton Dickinson, Sparks, MD). In
those cases, approximately 2–4 mm2of mycelium was asep-
tically removed from the growing edge of the colony and
extracted with the UltraClean Plant DNA Isolation Kit (MoBio
Laboratories, Inc., Solana Beach, CA) as per the manufactur-
Primers ITS1-F (fungal-specific) (Gardes & Bruns 1993) and
ITS4 (White et al. 1990) were used for both PCR-amplification
and sequencing of the internal transcribed spacer region (ITS)
of the nuclear rDNA repeat for each isolate. PCRs were done in
25 mL reaction volumes with 12.5 mL of PCR Master Mix
(Promega Corp., Madison, WI), 1.25 mL each of 10 mM primers,
and 10 mL of diluted (10- to 100-fold) DNA template. Amplifi-
cation was done with an initial denaturation step of 5 min at
94?C; 35 cycles of 30 s at 94?C, 45 s at 50?C, and 45 s at 72?C;
and a final extension of 7 min at 72?C. PCR products were
purifiedwith Montage PCRCentrifugal FilterDevices
(Millipore Corp., Billerica, MA) according to the manufactur-
er’s protocol. Elongation factor-1 alpha (TEF) was also ampli-
fied for clavicipitaceous isolates 57 and 59 as described by
Rehner& Buckley (2005). Purified
sequenced with BigDye Terminator sequencing enzyme v.3.1
(Applied Biosystems, Foster City, CA) using 2 mL of diluted
BigDye in a 1:3 dilution of BigDye:dilution buffer (400 mM Tris
pH 8.0, 10 mM MgCl2), 0.3 mL of 10 mM primer, 10–20 ng of
cleaned PCR template, and H2O to 5 mL total reaction volume.
Cycle sequencing parameters consisted of a 2 min denatur-
ation step at 94?C, then 35 cycles of 94?C for 39 s, 50?C for
15 s, and 60?C for 4 min. Reaction products were cleaned by
ethanol precipitation and sequenced on an ABI 3100 Genetic
Analyzer (Applied Biosystems, Foster City, CA). Sequencing
reads were edited and contiguous sequences for each isolate
were assembled and edited in Sequencher v.4.1.4 (Gene Codes
Corp., Ann Arbor, MI).
To assign genotype groups, sequences from all 843 isolates
were compared to each other in Sequencher at the 99 %
homology level. Each contig was then edited by eye to remove
any prior editing errors, disassembled, and a final assembly of
all sequences was conducted at the 100 % homology level to
yield a total of 257 unique sequences. DNA sequences have
been deposited in GenBank; in most cases, only one sequence
was deposited when different isolates yielded identical
sequences (Table 1). Diversity and similarityindices(following
Arnold et al. 2003, Arnold & Lutzoni 2007) were calculated
using genotype groups as operational taxonomic units.
Diversity was calculated using Fisher’s alpha, which is robust
to differences in sampling intensity (see Arnold et al. 2007).
Similarity indices included two indices based on presence/
absence data only (Jaccard’s index, Sørensen’s index), and one
based on abundance data (Morisita–Horn index). All indices
were calculated using nonsingleton genotypes only (geno-
types recovered more than once) and range from 0 (no simi-
larity) to 1 (full similarity of endophyte communities).
Identification of isolates
Initial identification of all sequences was obtained by BLAST
analysis using BLASTn (http://www.ncbi.nlm.nih.gov/BLAST).
Penicillium and Aspergillus species were then further identified
(S.W. Peterson, unpublished). Exact matches to the sequences
from ex-type cultures were considered reliable identifications
for these genera. Where genealogical concordance multilocus
used to corroborate the identity of isolates (Taylor et al. 2000).
Identification of other isolates was derived by interpreting
a combination of the first 100 BLAST matches and the distance
tree results produced from BLAST-generated pairwise align-
ments. In general, taxonomic identification based on BLAST
was applied cautiously (i.e., at the genus level or above, and
with caution given the occurrence of misidentified sequences
in GenBank; see Vilgalys 2003; Arnold & Lutzoni 2007).
Sequences with high identity (98–100 %) to multiple isolates of
a given genus, and that also fell within that genus in distance
analyses, were assigned to that genus. Sequences with w92–
100 % identity to more than one genus within a single family,
and that also fell within that family in distance analyses, were
in our reference database
124 F.E. Vega et al.
Table 1 – Fungal endophyte genotypes isolated from various surface-sterilized, asymptomatic coffee tissues in Colombia, Hawai’i, Mexico and Puerto Rico . . . . . . . . . . . . .
(continued on next page)
Fungal endophyte diversity in coffee plants
Table 1 – (continued)
F.E. Vega et al.
(continued on next page)
Fungal endophyte diversity in coffee plants 127
Table 1 – (continued)
F.E. Vega et al.
(continued on next page)
Fungal endophyte diversity in coffee plants129
considered to belong to that family. Sequences that shared
orderwereidentified onlyattheordinal level.A fewsequences
sharing less than 85 % identity with other sequences from
multiple orders, or sharing higher identity to unidentified
Results and discussion
Despite the fact that the methodology used here is culture-
dependent and slow growing and nonculturable taxa are
unlikely to be isolated (Hyde & Soytong 2008), a high diversity
of fungal endophytes was obtained. Various coffee plant
tissues served as suitable substrata for a wide number of
Ascomycota and a few Basidiomycota (Fig 1). A total of
843 fungal endophyteswas isolated: 267 from Colombia(32 %);
393 from Hawai’i (46 %); 109 from Mexico (13 %); and 74 from
Puerto Rico (9 %) (Table 1, Fig 1). These resulted in 257 unique
ITS sequences, yielding the following number of genotypes for
each region: Colombia, 113; Hawai’i, 126; Mexico, 32; and
Puerto Rico, 40. The most common endophytes were species
of Colletotrichum (251 isolates yielding 40 genotypes), Fusarium
(177 isolates yielding 25 genotypes), Penicillium (128 isolates
yielding 14 genotypes and 11 species) and Xylariaceae
(62 isolates yielding 26 genotypes).
All endophytes recovered from Coffea spp. were members of
the Dikarya. Eighty-seven percent of endophyte genotypes
recovered here represented the Ascomycota, and were
distributed throughout the Pezizomycotina. The majority rep-
resented the Sordariomycetes (Hypocreales, Phyllachorales,
Diaporthales, Sordariales, and Xylariales), Eurotiomycetes
(Eurotiales), and Dothideomycetes (Pleosporales) (Fig 2). The
dominance of Sordariomycetes in these primarily tropical and
near-tropical samples corroborates the findings of Arnold
& Lutzoni (2007), who found that Sordariomycetes dominated
the endophyte communities of tropical plants. However, the
present study recovered a greater representation of Euro-
tiomycetes than other surveys of tropical plants (e.g., Lodge
et al. 1996; Arnold et al. 2003), suggesting that Coffea spp. may be
an important reservoir for the often ecologically and econom-
ically important Eurotiales. The most common endophyte was
Colletotrichum; it has also been reported as the most common
endophyte intropicalTheobromacacao(Arnoldetal.2003),and is
a commonly isolated endophytic genus in the tropics, having
been reported in Amomum siamense, Anacardium occidentale,
Citrus spp., C. arabica, Euterpe oleracea, Glycine max, Guarea gui-
donia, Ilex paraguariensis, Malus domestica, Musa acuminata,
Himatanthus sucuuba, Palicourea longiflora, Strychnos cogens,
Spondias mombin, Zea mays, and other species (Bussaban et al.
et al. 2002; Arnold et al. 2003; Photita et al. 2004; Camatti-Sartori
et al. 2005; Rubini et al. 2005; Arnold 2007; Huang et al. 2008).
Basidiomycota, but these were distributed across all three
subphyla – Pucciniomycotina, Ustilaginiomycotina (Exobasi-
diomycetes: Exobasidiales and Tilletiales; and Ustilaginomy-
cetes: Ustilaginales) and Agaricomycotina (Tremellomycetes:
Tremellales; and Agaricomycetes:Agaricales and incertae sedis),
with the majority belonging to the Agaricomycetes (Fig 2).
Table 1 – (continued)
130F.E. Vega et al.
Although endophytic basidiomycetes are relatively rarely
reported (Petrini 1986, Petrini et al. 1992), some have been
recovered from temperate trees (e.g., Marasmius, Rhizoctonia,
Rhodotorula; Petrini & Mu ¨ller 1979; Widler & Mu ¨ller 1984;
Sieber-Canavesi & Sieber 1987, Arnold et al. 2007) and tropical
trees (e.g., Coprinellus, Fomitopsis, Phanerochaete, Pycnoporus,
Schizophyllum, Sebacina; Crozier et al. 2006; Rungjindamai et al.
2008; Tao et al. 2008).
Genotypic richness and diversity differedamong countries,
ranging from 32 genotypes among 109 isolates in Mexico
Fig 1 – Total number of fungal endophytes isolated in each country grouped by coffee plant tissues from which they were
isolated. Numbers above bar represent percentages.
Ascomycota incertae sedis
Basal fungal lineages
incertae sedis (5)
Incertae sedis (2)
Fig 2 – Schematic representation of phylogenetic placement of 257 fungal genotypes derived from ITS sequences of
endophytes isolated from coffee plants in Colombia, Hawai’i, Mexico, and Puerto Rico. Classification follows Hibbett et al.
(2007). All 257 genotypes represent the crown fungal group Dikarya (Basidiomycota plus Ascomycota). Isolates were
distributed among all three subphyla of Basidiomycota, but in only one of the three subphyla of Ascomycota
(Pezizomycotina). Percentages indicate the total number of genotypes out of 257 for indicated group; total number of unique
genotypes per taxonomic group is indicated in parentheses.
Fungal endophyte diversity in coffee plants131
(Fisher’s alpha¼14.9) to 113 genotypes among 267 isolates in
Colombia (Fisher’s alpha¼ 75.3) (Table 2). Mean diversity of
fungi recovered from Coffea tissues in Puerto Rico, Hawai’i,
and Colombia was significantly greater than in Mexico
(Table 3). Inclusion of additional Coffea spp. in Hawai’i did not
notably increase diversity of fungi recovered there, as all
genotypes recovered from other Coffea species also were
found in C. arabica (Table 1). Moreover, inclusion of multiple
sites within a country (e.g., Mexico) did not strongly increase
diversity relative to countries with only one thoroughly
sampled site (e.g., Colombia) (Tables 1, 2). Although Fisher’s
alpha is robust to differences in sample size, more isolates
from Mexico and Puerto Rico, sampling the same tissues in
each country, and explicit evaluation of local microclimate
conditions – which can strongly affect endophyte diversity
(e.g., Hoffman & Arnold 2007) – would increase our confidence
in the observed differences in diversity among countries.
Overall, only 107 genotypes (41.6 %) were recovered more
than once. Of these, 70 genotypes (65.4 %) were found in only
one country. Among the remaining 37 genotypes, 25 were
found in two countries, 10 were found in three countries, and
two were found in all four countries (Table 4). Genotypes
found in three or more countries represented only three
genera (Colletotrichum, seven genotypes; Fusarium, three
genotypes; Penicillium, two genotypes). These commonly
isolated genera also included genotypes found in two coun-
tries (Colletotrichum, five genotypes; Fusarium, three genotypes;
Penicillium, six genotypes); genotypes found in only one
country (Colletotrichum, 11 genotypes; Fusarium, 14 genotypes;
and Penicillium, four genotypes). Genotypes that were found in
at least two countries represented Aspergillus, Beauveria,
Botryosphaeria, Cladosporium, Colletotrichum, Fusarium, Mycos-
In contrast, none of the clavicipitaceous, pleosporaceous,
xylariaceous genotypes was found in more than one country,
and several genera were found only in one country’s coffee
plants (e.g., Clonostachys, Petriella, Tilletia, Trichoderma; Table 1).
Despite the occurrence of some genotypes in multiple
sites, similarity indices based on presence/absence and
abundance data showed low similarity among the fungal
communities recovered in different countries. Hawai’i and
SO¼0.368) and Colombia and Mexico (JI¼0.210, SO¼0.347)
(JI ¼0.226, whereasColombia
shared the most genotypes (Table 5). These results were
partially corroborated by abundance data, which showed the
greatest similarity between fungal assemblages in Colombia
(MH¼0.299; Table 5). Genotypes shared between Colombia
and Hawai’i represented Aspergillus, Beauveria, Botryosphaeria,
Cladosporium, Neosartorya, Trametes, and several genotypes
of Colletotrichum, Fusarium and Penicillium (Tables 1, 4). Geno-
types shared between Colombia and Mexico represented
Paecilomyces and the most widely distributed genotypes of
Colletotrichum, Fusarium, and Penicillium (Tables 1, 4).
The reasons underlying the especially wide distribution of
some Colletotrichum, Fusarium, and Penicillium genotypes
recovered here remain to be explored. It is possible that these
genotypes are ubiquitous among coffee-growing regions
because of intrinsic factors (i.e., global distribution of the
fungi themselves) or because of the movement of Coffea plants
and seeds, and their attendant endophytes, among coffee-
growing regions. Notably, 55 % of genotypes recovered from
seeds were found in multiple countries (Table 1). These data
may hint that some genotypes were distributed with seeds to
new locations, but this hypothesis needs further in-depth
research. The occurrence of some genotypes in leaves and
other tissues from multiple countries leaves open the possi-
bility of global distributions of fungi. Future studies would
benefit from a phylogenetic perspective that could explicitly
trace the biogeographic or colonization patterns of particular
When Hawai’ian endophytes are divided by islands of
origin (i.e., Oahu, Hawai’i, and Kauai) only 15 genotypes were
shared by two islands. Of these, 14 were shared between Oahu
and Hawai’i (Table 1). Only three endophytes (Colletotrichum
sp. 24, Colletotrichum sp. 28, and Penicillium olsonii) were
shared among the three islands. We found no evidence that
endophyte communities differed markedly among Coffea
species (Table 1), suggesting that endophyte communities are
influenced more by site than by plant host species per se. This
site-specific trend has been previously reported (Petrini 1985;
Herreetal. 2005;Santamarı ´a& Bayman2005; seealsoHoffman
& Arnold 2007). In another perennial tropical crop, T. cacao,
Arnold et al. (2003) reported a reduction in similarity among
endophytes as distance between sampling sites increased
>50 km. Santamarı ´a & Bayman (2005) also reported significant
differences in coffee fungal endophytes in Puerto Rico across
Table 2 – Sampling effort, genotypic richness, total diversity (Fisher’s alpha), and dominant genera and genotypes of fungal
endophytes recovered from surface-sterilized coffee tissues in Colombia, Hawai’i, Mexico, and Puerto Rico
(isolates, tissue type)a
Colletotrichum (106, 25)
Colletotrichum (121, 20)
Fusarium (68, 8)
Fusarium (24, 8)
Colletotrichum sp. 2 (25; L,C,P,St)
Penicillium olsonii (32; L,B,C,P,Se)
Fusarium sp. 16 (48; L,B,C,P,Se)
Fusarium sp. 18 (8; B,C);
Fusarium sp. 2 (8; B,C,P,Se)
Total843257125.9Colletotrichum (251, 40)Fusarium sp. 16 (64; L,B,C,P,Se,St,R)
a Tissue types: L, leaf; B, berry; C, crown; P, peduncle; Se, seed; St, stem; R, root.
132F.E. Vega et al.
Even though it has been suggested that endophytes in the
leaves of woody plants are acquired from air spora in the
environment (Petrini 1991; Arnold & Herre 2003), our results
indicate that vertical transmission might also be possible
based on the isolation of several endophytic fungi from coffee
seeds in all the countries sampled (Table 1) as well as from
green coffee seeds examined in a previous study (Vega et al.
2008a). Endophytic fungi have been reported in seeds of
Pseudotsuga menziesii (Bloomberg 1966), Casuarina equisetifolia
(Bose 1947), and Cecropiaspp. (Gallery et al. 2007). All genotypes
recovered from seeds in our study were also found as endo-
phytes in other tissues (Table 1).
In the present study diversity of fungi associated with
different tissue types did not differ significantly (ANOVA;
F6,12¼0.7746, P¼0.6048), although diversity was nearly seven-
fold higher in leaves (Fisher’s alpha mean?SEM¼31.1?8.6)
than in seeds (Fisher’s alpha¼ 4.6?2.2) (Table 3). The vast
majority of nonsingleton genotypes (75 genotypes, or 70 % of
those found more than once) were recovered from more than
one tissue type (e.g., leaves, stems, roots); the remaining 32
nonsingleton genotypes were found in only one tissue type.
Among the genotypes found only in one tissue type, 15 were
found only in leaves, three only from berries, four only from
crowns, five only from peduncles, two only from roots, and
three only from stems (Table 6).
Most of those single-tissue genotypes (28 of 32) were
isolated from a given tissue type in only one country.
However, four genotypes found only in leaves were recovered
from foliage in multiple countries (Table 6). These fungi may
be especially interesting for further study: on the one hand,
they may have global distributions and simply represent
opportunistic infections by generalist fungi. Alternatively,
they may represent closely co-evolved endophytes of Coffea
that have moved with plants across the coffee-growing
regions of the world.
In several cases, endophytes associated with only one
tissue type in one locality were subsequently found in
additional tissue types in other countries. For example,
Colletotrichum sp. 2 was recovered from leaves in Mexico, but
from crown, peduncle, stem, and leaf tissue in Colombia
(Table 1). In several cases, genotypes that would have been
assigned to singleton genotypes in survey of only one site or
tissue type were shownto be quite common in other tissuesor
sites, underscoring the need to broadly sample different
tissues and sites to adequately address the frequency and
tissue specificity of endophytic fungi (Table 1).
Overall, 63 of the 257 unique genotypes (including
singletons) were isolated only from leaves. It is likely that
these fungi, as has been reported for woody endophytes in
general, do not have the capacity to move through the plant
and only occur locally relative to the point of entrance
(see Saikkonen et al. 1998, Herre et al. 2007). The fungal species
found in leaf tissue, like most tissues, could reflect the
prevalence of aerial spores at a particular site, the ability of
sporesof individual species to growintothe leaf,and presence
offavorableconditionsthatallow ambientmicrofungi to enter
internal plant tissues.
Widler & Mu ¨ller (1984), in what to our knowledge is the
most comprehensive analysis of fungal endophytes in one
plant species, reported more than 190 different fungal
endophytes intheleaves,roots,and branchesofArctostaphylos
uva-ursi in two locations in Switzerland. Our coffee survey
surpasses this figure, with a total of 257 genotypes recovered.
While these genotypes do not correspond to species – indeed,
they overestimated species boundaries for Penicillium – our
data provide a fine scale of resolution for determining the
occurrence of endophytes among different tissue types, Coffea
spp., and geographic regions.
The high biodiversity of fungal endophytes in coffee
plants may indicate that most of these are ‘‘accidental
Table 3 – Mean and standard error of the mean for diversity (Fisher’s alpha) of fungal endophytes as a function of tissue
type (panel A) and country (panel B). Within each panel, means with the same numerical subscripts do not differ
Standard error of mean
a Data are included if ?10 isolates were recovered and sequenced from a given tissue in a given country. Data from crown samples in Hawai’i
were excluded because the diversity value, reflecting the recovery of 24 genotypes from 25 crown samples, was more than three standard
deviations greater than the mean.
b Statistical analyses reflect ANOVA with alpha ¼ 0.05, followed by post-hoc comparisons.
c Statistical analyses reflect ANOVA with alpha ¼ 0.05, followed by post-hoc comparisons; F3,15¼4.4682, P¼ 0.0197.
Fungal endophyte diversity in coffee plants 133
Table 5 – Number of nonsingleton genotypes (genotypes that occurred more than once), percent of all genotypes occurring
more than once, and similarity of fungal communities recovered from coffee tissues in Colombia, Hawai’i, Mexico, and
Puerto Rico, considering presence/absence data (Jaccard’s index, JI; Sørensen’s index, SO) and abundance data
(Morisita–Horn index, MH)
Country 1Country 2 Nonsingleton genotypes
country 1 (%)
country 2 (%)
52 (46.0 %)
52 (46.0 %)
52 (46.0 %)
62 (49.2 %)
62 (49.2 %)
23 (71.8 %)
62 (49.2 %)
23 (71.8 %)
21 (52.5 %)
23 (71.8 %)
21 (52.5 %)
21 (52.5 %)
Approximately half of all genotypes found in Colombia, Hawai’i, and Puerto Rico were found only once; in contrast, the majority of genotypes
recovered in Mexico were found more than once.
Table 4 – Numberof isolates of fungal endophyte genotypes that were recoveredfrom at least two countries among the four
sampled (Colombia, Hawai’i, Mexico, and Puerto Rico)
Genotype Colombia Hawai’iMexico Puerto Rico
Agaricomycetes sp. 1
Aspergillus sp. 3
Cladosporium sp. 3
Colletotrichum sp. 2
Colletotrichum sp. 4
Colletotrichum sp. 5
Colletotrichum sp. 7
Colletotrichum sp. 11
Colletotrichum sp. 20
Colletotrichum sp. 21
Colletotrichum sp. 23
Colletotrichum sp. 24
Colletotrichum sp. 25
Colletotrichum sp. 28
Colletotrichum sp. 29
Fusarium sp. 2
Fusarium sp. 4
Fusarium sp. 6
Fusarium sp. 13
Fusarium sp. 16
Fusarium sp. 18
Paecilomyces sp. 2
Penicillium sp. 1
Penicillium sp. 2
Penicillium sp. 9
Phomopsis sp. 17
A total of 843 endophytes was isolated, from which 257 unique ITS sequence genotypes were identified; of these, 220 genotypes were only found
in one country; 25 genotypes were shared by two countries; 10 genotypes were shared by three countries; and two genotypes were shared by
134 F.E. Vega et al.
tourists’’ with no role in the plant, in contrast to endophytes
that could be defined as ‘‘influential passengers’’ and whose
role on the plant has been elucidated. Using ribosomal DNA
sequence comparisons, Promputtha et al. (2007) suggested
that endophytic Colletotrichum, Fusarium, and other taxa in
Magnolia liliifera can change their lifestyle and become sap-
rotrophic after host senescence. Such ‘‘lifestyle switching’’
(Rodriguez & Redman 2005) might help explain the possible
roles of some fungal endophytes. Similarly, most members of
the Xylariaceae (Ascomycota: Sodariomycetes) are consid-
ered to be saprotrophs (Petrini & Petrini 1985; Weber & Anke
2006), however, they are especially common as endophytes of
tropical hosts (Gamboa & Bayman 2001; Peixoto Neto et al.
2002; Takeda et al. 2003; Tomita 2003; Crozier et al. 2006) and it
is possible that these play a saprotrophic role in coffee and
other plants after host senescence. The ecological roles of
Penicillium species remain to be explored and represent an
area of special interest to us for future studies. However,
experimental trials are needed to confirm the ecological roles
in living plants, or lack thereof, of the many fungi recovered
here (see Saikkonen et al. 2006 for possible roles). Notably,
Arnold et al. (2003) showed that resistance of T. cacao seed-
lings to invasion by a virulent pathogen (Phytophthora sp.)
occurred in the presence of multiple endophyte species in the
same leaf tissues. Similarly, Arnold & Lewis (2005) reviewed
several cases in which the entomopathogen B. bassiana was
able to protect host plants against a significant herbivore
even in the context of additional fungal inhabitants of the
The large number of singletons recovered here suggests
that we have barely scratched the surface of the diversity of
endophytes associated with Coffea. In particular, sampling
these plants in their biogeographic regions of origin would
elucidate the ways in which introduction to novel environ-
ments change the fungal communities with which economi-
cally important plants associate. Hoffman & Arnold (2007)
showed that trees in the Cupressaceae, when cultivated in
non-native environments, maintained a lower diversity of
fungi than did closely related, native species. Moreover, the
introduced species examined in that study consistently
harbored more cosmopolitan, less-specific endophytes than
did their native relatives in the same environments. The
movement of Coffea throughout the coffee-growing regions of
the world provides a useful framework for addressing similar
questions in an economically important plant in the fungus-
rich tropics. Furthermore, if other molecular techniques (e.g.,
DNAcloning:Guo etal. 2000,2001; Seena et al. 2008; denaturing
gradient gel electrophoresis (DGGE): Nikolcheva et al. 2003;
Nikolcheva and Ba ¨rlocher 2004, 2005; Duong et al. 2006; Tao
et al. 2008; or terminal-restriction fragment length poly-
morphism (T-RFLP): Nikolcheva et al. 2003; Nikolcheva &
Ba ¨rlocher 2005) were applied to identify fungal DNA from
leaves or other parts of the coffee plant, many slow growing
or unculturable fungi could be identified. A microarray
Table 6 – Fungal endophytes of Coffea spp. recovered from only one tissue type: genotype identification, tissue, and country
Genotype Tissue Countries of origin
Aspergillus sp. 4
Cladosporium sp. 5
Clonostachys cf. rosea
Colletotrichum sp. 5
Colletotrichum sp. 12
Colletotrichum sp. 16
Colletotrichum sp. 20
Colletotrichum sp. 22
Colletotrichum sp. 29
Colletotrichum sp. 30
Colletotrichum sp. 32
Colletotrichum sp. 36
Colletotrichum sp. 40
Fusarium sp. 19
Fusarium sp. 5
Fusarium sp. 7
Fusarium sp. 9
Phomopsis sp. 13
Tilletia sp. 1
Trichoderma sp. 1
Xylariaceae sp. 15
Colombia, Hawai’i, Mexico
Colombia, Hawai’i, Mexico
Fungal endophyte diversity in coffee plants 135
hybridization technique known as PhyloChip, currently used
for the identification of archaeal and bacterial organisms
(Brodie et al. 2007; DeSantis et al. 2007) is currently being
developed for the identification of fungal diversity (M. Black-
well, pers. comm.). This technique will greatly enhance our
understanding of fungal endophyte communities. We expect
that many more fungal endophytes in coffee remain to be
identified. Future research will focus on fungal endophyte
biodiversity in Africa, and on the potential applications of
these phylogenetically diverse and species-rich fungal asso-
ciates of Coffea plants.
FEV wishes to express his most sincere appreciation to
C. Nagai,S. Bittenbender, B. Sipes, D.R. Ching, V. Easton Smith,
A. Teramura, R. Baker, T. Martin, R. Loero, D.W. Orr, G. Staples,
R.A. Franqui, E.H. Otero, and C. Quintero for their assistance in
the field. Special thanks to K. Hyde for his comments on
a previous version of this paper. The use of trade, firm, or
corporation names in this publication is for the information
and convenience of the reader. Such use does not constitute
an official endorsement or approval by the United States
Department of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of others
that may be suitable.
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