American Journal of Plant Sciences, 2013, 4, 917-921 Published Online April 2013 (
Endophytic Mycobiota Characterization of the Amazonian
Mistletoe Cladocolea micrantha Hos ted in Cashew Tree
Anderson C. Guimarães1, Antonio C. Siani2*, José L. Bezerra3, Antonia Q. Lima de Souza4,
Maria Inez M. Sarquis5
1Instituto de Ciências Exatas e Tecnologia de Itacoatiara, Universidade Federal do Amazonas, Itacoatiara, Brazil; 2Departamento de
Produtos Naturais, Farmanguinhos, Fiocruz, Rio de Janeiro, Brazil; 3Departamento de Ciências Agronômicas e Ambientais,
Universidade Estadual de Santa Cruz, Ilhéus, Brazil; 4Laboratório de Genética, Escola Superior de Ciências da Saúde, Universidade
Estadual do Amazonas, Manaus, Brazil; 5Laboratório de Taxonomia Bioquímica e Bioprospecção de Fungos, Instituto Oswaldo Cruz,
Fiocruz, Rio de Janeiro, Brazil.
Email: *
Received March 5th, 2013; revised April 3rd, 2013; accepted April 12th, 2013
Copyright © 2013 Anderson C. Guimarães et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Endophytic fungi were identified from different parts of the medicinal parasitic mistletoe Cladocolea micrantha and
from its host Anacardium occidentale, suggesting a strict host-parasite relationship. Eight fungal endophytes were iso-
lated and morphologically characterized. The ascomycete Guignardia mangiferae and strains of Mycelia sterilia were
prevalent in the isolations. The unequivocal identification of Guignardia mangiferae at a probabilistic degree close to
100% was carried out by DNA extraction followed by PCR analyses of the ITS-1 and ITS-2 regions and comparison of
the genetic sequence with the NCBI database.
Keywords: Cladocolea micrantha; Endophytic mycobiota; Loranthaceae; Guignardia mangiferae; Anacardium
1. Introduction
The Loranthaceae mistletoes, popularly known in Brazil
as “erva-de-passarinho” [1] play an important and com-
plex part in the biological system they inhabit, interacting
with insects, birds, fishes and mammals [2]. These epi-
phytic and hemiparasitic plants adhere to the branches
and twigs of trees by means of haustoria, penetrating the
host to absorb water and nutrients [3]. Species of this
family are used all over the world in traditional medicine
to treat a wide range of diseases, such as arthritis, diabe-
tes, general inflammation, breathing and nervous prob-
lems and some types of cancer [4]. In the Amazonian
region, the leaves of the species Cladocolea micrantha
(Eichler) Kuijt are widely used in the non-conventional
therapy of several types of tumors and inflammatory
processes [5], with a preference for specimens growing
on the cashew tree Anacardium occidentale (Anacardi-
aceae) [6]. The genus Cladocolea comprises approxi-
mately 40 species and occurs from central to southern
Mexico and in some localities of Latin America. The
most important morphological feature of this genus is the
occurrence of a determinate inflorescence and single lat-
eral ebracteolate flowers or a simple derivative thereof;
this characteristic serves to differentiate Cladocolea from
other similar mistletoe genera [7]. Chemical studies on
this genus are restricted to Cladocolea micrantha which
describes the isolation of diverse triterpenes as well as
kaempferol and quercetin derivatives presenting an un-
common interglycosidic O-(13) linkage [8]. Comple-
mentarily, the triterpenes α-amyrin, β-amyrin and their
respective ketones were remarkably characterized from
the chloroform extracts of mycelia of G. mangiferae, P.
clavisporus and P. guepinii 7-d to 14-d-old colonies, iso-
lated from this mistletoe [9].
Endophytic fungal communities occupy intercellular
organs in plants, usually inducing an associative rela-
tionship that ranges from mutualism to host plant para-
sitism. Such a symbiosis may confer some protection
against insects, herbivores or phytopathogenic microor-
ganisms by producing toxins, antibiotics, specific en-
zymes or growth-promoting agents and other substances
[10]. Endophytic fungi have also been found to be even-
*Corresponding author.
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Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree
tually responsible for the bioactive compounds produced
by host plants. A corollary to this assumption would be
the production of the anti-cancer agent taxol by the
endophytic fungi Taxomyces andreanae, Pestalotiopsis
microspora and P. guepinii isolated from the Pacific
Taxus brevifolia. Similarly, the fungus Phialocephala
fortinii isolated from Podophyllum peltatum may pro-
duce the antitumoral podophyllotoxin [11,12]. Endo-
phytes have been identified on species associated with
many botanical families, and currently, a significant
amount of research is focused on obtaining active me-
tabolites from endophytes, aiming to utilize them in
medicine and biological control [11,13,14]. The present
study aimed to identify and to morphologically and mo-
lecularly characterize the endophytic fungi from different
parts of the parasite mistletoe Cladocolea micrantha and
from its host Anacardium occidentale.
2. Material and Method
2.1. Plant Material
Leaves and stems of Cladocolea micrantha (Eichler)
Kuijt and Anacardium occidentale L. for the host-para-
site mycobiotic analyses were collected on the campus of
the Federal University of Amazonas (UFAM), Manaus,
Brazil, S 03˚07.785', W 058˚26.631', each from a single
individual host specimen 8 m tall. Both species were
identified, and vouchers were deposited in the Herbarium
of UFAM under No. 6213.
2.2. Surface-Disinfecting and Inoculation of the
Botanic Material
Endophytic fungi were isolated according to the proce-
dure by Petrini (1992) [15,16] using fresh botanical ma-
terial from both C. micrantha and A. occidentale. The
plant material was carefully washed with a neutral deter-
gent and running water. After drying, the leaves and
stems were sliced into pieces of approximately 8 cm2 and
were soaked for 1 min in a 500 ml beaker containing
70% ethanol in water. The fragments were then trans-
ferred to another beaker containing a 3% solution of so-
dium hypochlorite for a period of 4 min. The pieces of
leaves and stems were then returned to the beaker con-
taining the ethanol/water solution for 30 s and were fi-
nally transferred to a flask with sterilized water for 1 min.
After this procedure, fragments of leaves and stems from
both plants were cultured on eight Petri plates (six frag-
ments per plate) containing malt-agar extract (MAE)
(Difco) and chloramphenicol (100 μg/ml MAE) with the
aid of a Drigalski loop, resulting in 192 inocula distrib-
uted onto 32 plates. A negative control was run by ap-
plying 0.05 ml of the distilled water from the last sterili-
zation step onto Petri dishes using the same medium.
Cultured samples were incubated at room temperature
(approximately 25˚C in the laboratory) and observed
2.3. Endophytic Fungi Isolation and
After eleven days, the plates contained 160 growing
colonies that were singly prepared on slides and carefully
inspected under a microscope. Identical fungal strains
were separately pooled into eight different representative
groups according to their macro- and micromorphologi-
cal similarities, and their purity was certified by macro-
and microscopic evaluation. For genus and species as-
signments, the fungal strains were separately grown on
the following media: PDA, malt agar extract (Difco),
Czapek-dox agar (Merck) and oatmeal agar (60.0 g oat-
meal and 18.0 g agar). Nutrient-poor synthetic medium
agar [1.0 g KH2PO4, 1.0 g KNO3, 0.5 g MgSO4·7H2O,
0.5 g KCl, 0.2 g glucose, 0.2 g sucrose and 18.0 g agar
(all Merck) in 1000 ml distilled water] and sunflower
seed extract agar were also used according to classical
methodologies [17]. The microscopic characteristics were
evaluated in microcultures on coverslips in a drop of
Amann’s lactophenol [20.0 g phenol (Quimibras), 20.0
ml lactic acid (Merck), 20.0 ml distilled water, 40.0 ml
glycerine (Merck)] with 0.05 g cotton blue (Sigma). The
filamentous fungi were classified based on standard pro-
cedures reported for morphological characterization [18-
20]. The fungi were then transferred to test tubes con-
taining MAE for successive growth. Samples were ly-
ophilized and stored under mineral oil in the Fungi Cul-
ture Collection Filamentous Fungi of the Oswaldo Cruz
Institute, Rio de Janeiro, under Nos. IOC4553 (Aspergil-
lus sp.), IOC 4548 (G. mangiferae), IOC4555 (M. ster-
ilia), IOC4549 (Myrothecium sp.), IOC4550 (P. clavis-
porus), IOC4551 (P. guepinii), IOC4552 (T. pseudoviri-
dae) and IOC4554 (Drechslera sp.).
2.4. DNA Extraction and PCR Conditions
G. mangiferae identification was confirmed by sequenc-
ing the ITS-1 and ITS-2 rDNA and comparing it with
sequences from GenBank. For ITS-1 and ITS-2 rDNA
analyses, the strain was stirred at room temperature for
12 days (120 rpm) on a potato-dextrose medium enriched
with 0.2% PDY (potato dextrose yeast) extract. From
200 mg of mycelium separated by filtration, genomic
DNA was extracted using the cetyltrimethylammonium
bromide method [21]. Prior to PCR, the DNA concentra-
tion was visually estimated at 100 ng/μl by comparing it
with the 1 kb marker on a 1% agarose gel. Each 25 μl of
reaction contained 10.2 μl of water, 2.5 μl of 10× buffer,
3.0 μl of 25 mM MgCl2, 3.0 μl of 1.25 mM dNTP, 2.0 μL
each of ITS-1 and ITS-2 primers at 10 mM, 0.3 μl of Taq
Copyright © 2013 SciRes. AJPS
Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree
Copyright © 2013 SciRes. AJPS
DNA polymerase (5.0 U) and 2.0 μl of genomic DNA.
Four reactions were carried out with an initial denaturing
step of 94˚C for 4 min, followed by 40 cycles of 2 min
denaturing at 94˚C, 2 min annealing at 55˚C and 2 min
elongation at 72˚C. The amplification was terminated for
10 min at 72˚C. PCR products were analyzed by electro-
phoresis on 1.5% agarose gels stained with ethidium
bromide, compared with a DNA ladder of 100 bp and
estimated at 50 ng/μl. In all, 2 μl of the PCR products
were sequenced on an acrylamide gel, and the sequences
obtained were matched with those deposited in the NCBI
(National Center for Biotechnology Information) data-
3. Results and Discussion
A total of 160 fungal strains (from the 192 total frag-
ments of plant material) comprising 8 groups of endo-
phytic fungi were isolated from both mistletoe and the
plant host. Stems and leaves of the mistletoe Cladocolea
micrantha led to the isolation of the following fungal
species and genera: Aspergillus sp. (Trichocomaceae,
Order Eurotiales), Guignardia mangiferae (Botryosphae-
riaceae, Order Botryosphaeriales) Mycelia sterilia (deu-
teromycetes, Order Agonomycetales), Myrothecium sp.
(Incertae sedis, Order Hypocreales), Paecilomyces clav-
isporus (Trichocomaceae, Order Eurotiales), Pestalo-
tiopsis guepinii (Amphisphaeriaceae, Order Xylariales)
and Trichoderma pseudoviridae (Hypocreaceae, Order
Hypocreales). The following species and genera were
identified in the tissues of Anacardium occidentale:
Pestalotiopsis guepinii, Drechslera sp. (Pleosporaceae,
Order Pleosporales), Guignardia mangiferae, Mycelia
sterilia, and Trichoderma pseudoviridae. The three spe-
cies/genera in boldface were common to both the parasite
and the host plant (Ta ble 1). All of these occurrences are
described here for the first time, especially for the
Cladocolea micrantha mycobiota, for which, as far as we
know, this is the first report.
The non-sterile ascomycetes G. mangiferae and Myce-
lia sterilia were found as the most frequent fungal endo-
phytes in both the parasite and the host plants in the ex-
perimental sampling. Identification of G. mangiferae was
based on macroscopic characteristics and on the micro-
scopic measurements of individuals from the 7-d-old
colony on MAE, which presented the following dimen-
sions: pseudothecia [(640 - 1232) × (112 - 424) μm];
ostioles [(80 - 400) × (80 - 200) μm]; asci [(40 - 68) ×
(10 - 12) μm; ascospores [(15 - 16) × (5 - 6) μm] [22].
Similar results for the ascospore sizes [(13 - 17) × (5 - 8)
μm] were observed for G. mangiferae isolated from Ana-
cardium giganteum [23]. The trusted pairwise compari-
sons revealed 99% identity with G. mangiferae and 100%
reliability for the 576 DNA base pairs (data not shown).
Such a high probabilistic correlation provides additional
relevant data on tropical endophytes, given that the Gui-
gnardia species present wide intraspecific genetic vari-
ability [24,25]. Species of Guigna rdia are usually recog-
nized as endophytic fungi and have been isolated from
asymptomatic tissues from a diversity of Myrtaceae and
Anacardiaceae species as well as citrus plants [25]. How-
ever, some species such as G. citricarpa and G. psidii
cause serious diseases to agriculture by infesting fruit
and leaves post-harvest. General studies of endophytes
are more prevalent in temperate regions [26] and should
be extended to tropical species when considering the
pharmaceutical potential of tropical medicinal plants [27].
4. Conclusion
The findings reported here reinforce the cosmopolitan
occurrence of the endophytic G. mangiferae in tropical
plants [23,28,29]. The non-sterile mycobiota of the leaves
of both parasitic and host plants is characterized by the
common presence of G. mangiferae as the most frequently
occurring fungus. This presence was not observed for the
Table 1. Endophytic fungi characterized in the tissues of Cladocolea micrantha and Anacardium occidentale.
Fungus species CM leaf CM stem AO leaf AO stem
Aspergillus sp. [2] n n 2/10 n
Drechslera sp. [1] n n 1/10 n
Guignardia mangiferae [15] 8/15 n 7/10 n
Myrothecium sp. [2] 1/15 1/18 n n
Mycelia sterilia [133] 31/15 38/18 35/10 29/3
Paecilomycesclavisporus [2] 2/15 n n n
Pestalotiopsis guepinii [2] n 2/18 n n
Trichoderma pseudoviridae [3] 1/15 n 1/10 1/3
CM = Cladocolea micrantha; AO = Anacardium occidentale. In brackets = total of similar pooled strains. Results (X/Y) indicate the ra-
tio of culture plates (X) growing positively over the total cultured plates (Y), including replication experiments; n = no growth observed.
Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree
stems, suggesting that some horizontal fungal contami-
nation (leaf-leaf) cannot be excluded [30]. This is the
first description of G. mangiferae as an endophyte in C.
micrantha. Moreover, its presence on A. occidentale sug-
gests a close host-parasite relationship, a fact that may be
relevant to the medicinal properties of the parasite plant
and to the popular preference for G. mangiferae grown
on this host plant.
5. Acknowledgements
The authors wish to thank Dra. Lilian A. Procópio for the
plant identification, and plant collectors Silo Silva and
Rosalba Bilby for their help.
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