American Journal of Plant Sciences, 2012, 3, 1105-1114
doi:10.4236/ajps.2012.38133 Published Online August 2012 (http://www.SciRP.org/journal/ajps)
1105
Diversity and Biological Activities of Endophytic Fungi
Associated with Micropropagated Medicinal Plant
Echinacea purpurea (L.) Moench
Luiz H. Rosa1, Nurhayat Tabanca2, Natascha Techen2, David E. Wedge3, Zhiqiang Pan3,
Ulrich R. Bernier4, James J. Becnel4, Natasha M. Agramonte4, Larry A. Walker2, Rita M. Moraes2,5
1
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Minas Gerais, Brazil;
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of
Mississippi, Oxford, USA; 3USDA-ARS, Natural Products Utilization Research Unit (NPURU), University of Mississippi, Oxford,
USA; 4USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), Gainesville, USA; 5Center for Water
and Wetland Resources, The University of Mississippi Field Station, Abbeville, USA.
Email: lhrosa@icb.ufmg.br, rmoraes@olemiss.edu
2
Received June 22nd, 2012; revised July 18th, 2012; accepted July 29th, 2012
ABSTRACT
Echinacea is one of the top ten selling medicinal herbs in Europe and United States. Commercially available formulations may contain different plant parts of three species (Echinacea purpurea, E. pallida, and E. angustifolia). Our study
evaluates the diversity of microbial community associated with healthy E. purpurea clones and their ability to produce
defense compounds. We recovered and identified thirty-nine fungal endophytes through the molecular methods in 15
distinct phylotypes, which were closely related to species of the following genera Ceratobasidium, Cladosporium Colletotrichum, Fusarium, Glomerella, and Mycoleptodiscus. These taxa were previously reported as decomposer and
phytopathogenic fungi. The fungal community associated with two E. purpurea clones showed high richness and
dominance indices with different distribution among plant organs. Crude extracts of fungal isolates were tested for antifungal and insecticidal biological activities. A total of 16 extracts (41%) showed antifungal properties; while just the
extract of M. indicus exhibited larvicidal activity against A. aegypti. These results suggest that the symbiosis between
the endophytic fungal community and micropropagated clones of E. purpurea was re-established after acclimatization
to soil and the endophytic fungi produced compounds against phytopathogenic fungi.
Keywords: Endophytes; Biological Activity; Medicinal Plant; Symbiosis; Botanical Drug; Microorganisms
1. Introduction
The role of symbiosis between plant and microorganism
is considered a key element for eukaryotes colonization
of the land [1]. Endophytic fungi have been recovered
from healthy tissues of plant species growing in different
biomes such as tundra, dry deserts, and tropical rainforests from the Arctic to Antarctica. These symbionts
produce metabolites that influence plant defense against
disease, prevent herbivores’ attack, while enhance growth
aiding the host’s survival under stressful conditions [2].
There are limited information on symbiotic relationship
between plants and endophytes to fully understand these
types of interactions. According to Taylor et al. [3], plants
removed from natural habitat are more susceptible to
pathogens attack due to a reduction of endophytes colonization.
Various studies have demonstrated that the endophytic
Copyright © 2012 SciRes.
fungal communities associated with medicinal plants
produce several bioactive compounds with different biological activities such as antimicrobial, cytotoxic, immunesuppressive and anti-parasitic to protect the host [46]. Echinacea is the second top-selling botanical supplement in the US market due to immune modulator properties [7]. Moraes et al. [8] established an in vitro repository of Echinacea sp. to produce healthy plants in an
effort to identify the active constituents responsible for
the immune enhancing activities. Pugh et al. [7] reported
differences in immune enhancing activities of Echinacea
shoot cultures, and later they demonstrated that monocyte and macrophage immune activation is due to lipoproteins and lipopolysaccharides (LPS) of bacterial endophytes. Lata et al. [9] detected endophytes associated
with in vitro shoot cultures, which were identified as
IAA producing bacteria. To further study the role of
endophytes in association with elite Echinacea purpurea
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Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
plants, we investigated the diversity of fungal endophytes
associated with micropropagated clones and their capabilities to produce defence compounds to protect its host
against pathogenic fungi and herbivores.
2. Material and Methods
2.1. Echinacea in Vitro Repository
The North Central Regional Plant Introduction Station
(NCRPIS), in Ames, Iowa provided seeds of E. purpurea
accession PI631307. All experiments were conducted
with shoot cultures obtained from hypocotyl explants.
Each germinated seed represented one clone. Seeds were
surface disinfected as follows: 1% NaOCl (20% v/v
bleach) and 0.1% Tween 20 for 10 min followed by
washing three times in sterile distilled water prior inoculation on the germination media. Aseptic explant initiated
shoots on half strength MS medium [10]. After 10 days,
0.5 - 1.0 cm long hypocotyls were taken as explants for
the initiation of shoot cultures. Shoots grew on half
strength MS salt medium containing 3% (w/v) sucrose,
0.8% (w/v) agar supplemented with 2.2 M of benzyladenine (BA) per liter. The medium was adjusted to pH
5.7. Shoot cultures have been maintained for 24 months
with transference each 30 days. All cultures were incubated at 25˚C ± 2˚C, 16-hours photoperiod under fluorescent light with a photon flux of 52 mol·m−2/s.
2.2. Plantlet Cultivation in Pots Maintained in
Greenhouse
Rooted plantlets of different clones of E. purpurea were
transferred to a soil substrate composed of a mixture (1:2
v/v) potting soil (Potting Mix Miracle Gro 0.14 0.14 0.14)
and sand (Garden Basic Play Sand, Sims Bark Co, Tuscumbia, AL). Potted plantlets were maintained under
mist-irrigation watering cycle for 1-min every hour during 6 hours period similar to the procedure reported for E.
pallida and E. angustifolia [9,11]. Two selected clones of
E. purpurea designated as PUR 02 and PUR 30 were
planted in pots maintained for three years. Endophytes
were isolated from these clones. Early springs plants
were fertilized with cottonseed meal 6-2-1 (The Espoma
Co, Millville, NJ).
2.3. Isolation of the Endophytic Fungi
Healthy leaves, lateral shoots, and roots of four plants of
each clones PUR 02 and PUR 30 maintained in greenhouse were cut into pieces, stored in plastic bags at 10˚C
for no more than 24 hours before isolation of the endophytic fungi. Different plant tissues were subjected to
surface sterilisation according to protocols established by
Rosa et al. [5]. The fragments were inoculated on Petri
Copyright © 2012 SciRes.
plates containing potato dextrose agar (PDA; Sigma/
USA) supplemented with chloramphenicol at 200 µg/ml
(Sigma/USA). The plates were incubated at 25˚C for up
to 30 days, and individual colonies were transferred to
PDA and stored at 4˚C. The fungal species were submitted to a long-term preservation for future research,
thus the mycelial pieces were stored in cryotubes with
30% sterilized glycerol at –80˚C.
2.4. Molecular Identification of Endophytic
Fungi
All endophytic fungi were identified by molecular methods. DNA from endophytic fungi was extracted with
DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and
used as template in PCR amplifications. The ITS1-5.8S
-ITS2 genomic region (ITS) was amplified from genomic
DNA using the forward primer ITS1 (5’-tccgtaggtgaacctgcgg-3’) and the reverse primer ITS4 (5’- tcctccgcttattgatatgc-3’) [12]. PCR amplifications were carried out in
50 μl reaction mixture containing 1 × PCR reaction
buffer, 0.2 mM dNTP mixture, 0.2 μM of each forward
and reverse primers, 1.5 mM MgSO4 and 2 U of Platinum Taq DNA Polymerase (Invitrogen, CA). The PCR
program consisted of one initial denaturation step at 94˚C
for 3 min followed by 40 cycles at 94˚C for 30 sec, 50˚C
for 30 sec, 72˚C for 1:30 min, with a final extension at
72˚C for 7 min. PCR were run in an M & J Research
Gradient Cycler PTC-225. After amplification, an aliquot
was analyzed by electrophoresis on a 1% TAE agarose
gel, visualized under UV light and PCR products were
compared to the molecular size standard 1 kb plus DNA
ladder (Invitrogen, CA). Successfully amplified PCR
products were extracted using MinElute PCR Purification Kit (Invitrogen, CA) and sequenced on an automated DNA Sequencer (model ABI 3730XL; Applied
Biosystems, Foster City, CA). Consensus sequence data
of the endophytic fungi were submitted to the GenBank
database (see accession numbers in Table 1). According
to Gazis et al. [13] the sequence of ITS regions may fail
to recognize some Ascomycota taxa; for this reason, the
following preliminary criteria were used to interpret the
sequences of the GenBank database: for sequence identities > 98%, the genus and species were accepted; for
sequence identities between 95% and 97%, only the
genus was accepted; for sequence identities < 95%, isolates were labelled as unknown species and identified in
family, class or order hierarchical levels [14]. However,
the phylotypes that displayed identities < 97% or inconclusive taxonomic level were submitted to phylogenetic
inferences, which were estimated using MEGA Version
5.0 [15]. The maximum composite likelihood model was
used to estimate evolutionary distance with bootstrap
AJPS
Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
1107
Table 1. Isolate code, number of isolates, closest related species, maximum identities, number of bp analyzed, identification,
and GenBank accession number of endophytic fungi phylotypes associated with Echinacea purpurea (L.) Moench.
a
UM
code
a
Part
N˚ of
isolates
01
L
1
15
L
43
Closest related species/GenBank accession
number
GenBank
accession
number
Maximum
identity (%)
N˚ of bp
analyzed
Ceratobasidium sp. [AB286937]
98
1638
Ceratobasidium sp.
HQ148092
8
Cladosporium cladosporioides (Fresen.)
G.A. de Vries [EU935608]
99
596
Cladosporium
cladosporioides
HQ148094
L
1
Colletotrichum gloeosporioides (Penz.) Sacc.
[AJ301979]
97
1189
Colletotrichum
gloeosporioides
HQ148099
32
L
6
C. gloeosporioides [GU810508]
98
598
C. gloeosporioides
HQ148097
71
R
1
Colletotrichum trifolii Bain &
Essary [AF451909]
95
1196
C. trifolii
HQ148103
55
R
1
Colletotrichum sp. [AB443952]
99
621
Colletotrichum sp.
HQ148100
68
R
1
Fusarium oxysporum Schltdl. [FJ156282]
100
773
Fusarium oxysporum
HQ148102
77
Sh
4
F. oxysporum [FJ157216]
99
495
F. oxysporum
HQ148104
34
Sh
2
F. oxysporum [FJ824032]
99
509
F. oxysporum
HQ148098
16
L
1
F. oxysporum [GQ365156]
92
565
Fusarium sp.
HQ148093
57
Sh
1
Fusarium solani (Mart.) Sacc. [AB518683]
98
612
F. solani
HQ148101
78
R
2
Fusarium sp. [GQ141219]
92
1462
F. oxysporum
HQ148105
95
1184
Glomerella sp.
HQ148106
1034
Mycoleptodiscus indicus
HQ148095
1138
Pleosporales sp.
HQ148096
b
90
L
1
Glomerella cingulata (Stoneman) Spauld.
& H. Schrenk [FJ172237]
28
S
3
Mycoleptodiscus indicus (V.P. Sahni)
B. Sutton [GU220382]
97
29
S
1
Pleosporales sp. [DQ092514]
90
b
Proposed identification
c
e
UM = Culture code of endophytic fungi; Leaf (L), Steam (S), Shoot (Sh), Root (R), and Tuber (T); Maximum identity; Fungi.
values calculated from 1000 replicate runs. Information
about the fungal taxonomic hierarchical levels follows
the databases MycoBank (www.mycobank.org) and Index Fungorum (www.indexfungorum.org).
2.5. Species Richness and Evenness Spatial
Analyses
For measurement of the species diversity, we used the
indexes: 1) Shannon H ni n ln ni n and 2)
Simpson’s = 1 sum ni n
2
, where ni is the number
of individuals of the taxon i and n is the total number of
individuals. All results were obtained with 95% confidence, and the bootstrap values were calculated from
1000 iterations. The index calculations were carried out
using the computer programme PAST version 1.90 [16].
2.6. Cultivation and Extraction of Fungal
Cultures
Five millimeter diameter plugs of each endophytic fungus were place at the center of Petri dishes (90 mm diameter), each containing 20 mL of PDA, and cultured for
13 days at 25˚C ± 2˚C [17]. The material from the resulting fungal cultures were cut from the each Petri dish
Copyright © 2012 SciRes.
and transferred to 50 mL vials tubes containing 25 mL of
ethanol. After 72 h at room temperature, the organic
phase was decanted and the solvent removed under vacuum centrifugation at 40˚C. An aliquot of the dried extract was dissolved in water/methanol (1:1) in order to
prepare a 20 mg/mL stock solution, which was stored at
–20˚C. A similar extraction procedure was carried out
using sterile PDA medium, and the extract obtained was
used as a control in the bioassays.
2.7. Biological Activities
The phytopathogens Colletotrichum acutatum, C. fragariae, and C. gloeosporioides were used as target species
for the antifungal assay. These microorganism were
grown on PDA (Difco, Detroit, MI) in 9 cm Petri dishes
and incubated in a growth chamber at 24˚C ± 2˚C under
cool-white fluorescent lights (55 ± 5 μmol/m2/s) with a
12 h photoperiod to sporulation. Conidia of three fungal
targets were harvested from 7 - 10 day old cultures by
flooding plates with 5 mL of sterile distilled water and
dislodging conidia by softly brushing the colonies with
an L-shaped plastic rod. Aqueous conidial suspensions
were filtered through sterile Miracloth (CalbiochemNovabiochem Corp., La Jolla, CA) to remove mycelia.
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Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
Conidia concentrations were determined photometrically
[18] from a standard curve based on absorbance at 625
nm, and suspensions were adjusted with sterile distilled
water to a concentration of 1.0 × 106 conidia/mL. Standard conidial concentrations were determined from a
standard curve for each fungal species. Standard turbidity
curves were periodically validated using both a Bechman/Coulter Z1 particle counter and hemocytometer
counts. Conidial and mycelial growth for microdilution
broth experiments were evaluated using a Packard model
SpectraCount microplate photometer (Packard Instrument Co., Meriden, CT). All extracts of endophytic fungi
were diluted in ethanol and spots at 80 and 160 µg
applied on the TLC plates. Each plate was subsequently
sprayed with a spore suspension (105 spores/mL) of the
target fungus of interest and incubated in a moisture
chamber for 4 days at 26˚C with a 12 h photoperiod.
Clear zones of fungal growth inhibition on the TLC plate
indicated the presence of antifungal activity in each
extract. Fungal growth inhibition was evaluated 4 - 5
days after treatment by measuring zone diameters. Antifungal metabolites were readily located on the plates by
visually observing clear zones where the active compounds inhibited fungal growth [19]. Spots of the fungicide technical grade benomyl (at 1.16 μg), cyprodinil
(at 0.9 μg), azoxystrobin (at 1.61 μg), and captan (at 1.2
μg) (Chem Service, Inc. West Chester, PA) diluted in
ethanol were used as standard controls. All antifungal
assays were performed in triplicate. All endophytic fungi
extracts were diluted in acetone to six concentrations
varying between 500 to 15.625 ppm and topically applied
to individual mosquitoes against Ae. aegypti. The procedure was performed according to description of Tabanca et al. [20].
3. Results
Thirty-nine endophytic fungi isolates were recovered,
which were closely related to the genera Ceratobasidium,
Cladosporium, Colletotrichum, Fusarium, Glomerella,
and Mycoleptodiscus (Table 1). Among the fungal community living within E. purpurea, Cladosporium cladosporioides, Colletotrichum gloeosporioides, and Fusarium oxysporum were the most abundant phylotypes,
while Ceratobasidium sp., Colletotrichum trifolii, Fusarium solani, Glomerella sp., and Pleosporales sp. Represented the scarce phylotypes within this community.
The phylogenetic trees of six phylotypes were constructed to illustrate the relationship of individual sequences to the closest relatives retrieved from GenBank
database. The ITS sequence of phylotype UM01 displayed a sequence identity of >96% to several sequences:
98% to Ceratobasidium sp. (AB286937), 98% to fungal
Copyright © 2012 SciRes.
endophyte sp. (FJ613838), 97% to Thanatephorus cucumeris (DQ223780), and 96% to Ceratobasidium cornigerum (EU273525). The sequence alignment (Figure
1(a)) of UM01 with the mentioned before sequences displayed only three nucleotides differences compared to
the ITS sequence of Ceratobasidium sp. (AB286937),
while more (eight) nucleotides difference were identified
when UM01 was compared to the ITS sequence of T. cucumeris (DQ223780). The phylotypes UM16 and UM78
displayed 92% identities with published ITS sequences
of Fusarium oxysporum. In the phylogentic analysis, the
phylotype UM16 presented 31 (5%) nucleotides differences to three strains of F. oxysporum (GQ365156,
FJ605243, and FJ605244). The phylotype UM78 showed
17 (2.7%) nucleotides differences with F. oxysporum
(HM179532) (Figure 1(b)). The phylotype UM29 displayed identity of 90% and 78 nucleotide difference
(13.6%) with Pleosporales sp. (DQ092514) and Didymella sp. (DQ092504) (Figure 1(c)). In addition, UM29
presented ≤ 90% sequence homology with several unidentified and uncultured fungi. The phylotype UM90
showed an identity of 95% with both the ITS sequence of
Glomerella cingulata (FJ172237) and Colletotrichum
gloeosporioides (FJ172224). The phylogenetic analysis
(Figure 1(d)) of UM90 displayed a 188 difference to C.
gloeosporioides (FJ172224) and a 189 nucleotide difference to G. cingulata (FJ172237). The phylotype UM55
showed a 100% nucleotide similarity with different Colletotrichum species. However, in the phylogenetic analysis, UM55 showed a high range of sequence identity
and only very few nucleotide differences [2 (0.3%) to 11
(1.9%)] (Figure 1(e)) with Colletotrichum sequences.
Three endophytes isolates were identified as Mycoleptodiscus indicus UM24, which displayed 97% of similarity
and only five nucleotides difference (0.8%) in compareson with different sequences of M. indicus deposited in
GenBank (Figure 1(f)). A total of four plants per two
clones E. purpurea were analysed about the associated
fungal community, which displayed high diversity (Shannon H = 2.4) and dominance (Simpson’s = 0.9). The
assemblage composition among the tissues of E. purpurea was variable (Table 1). Only the phylotype F.
oxysporum occurred in both plant tissues.
A total of 16 (41%) of the 39 endophytic fungi extracts
displayed antifungal activity against phytopathogenic
Colletotrichum species. Of the 16 endophytic fungi extracts, seven were strongly active against C. acutatum,
six displayed activity against C. fragariae, and eight demonstrated good activity against C. gloeosporioides
(Table 2). In addition, 10 phylotypes showed extracts
with fungicidal activities and six produce a diffuse halo
against the Colletotrichum targets. The Fusarium genus
was the most active with eight endophytes phylotypes.
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Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
1109
Rhizoctonia sp. [AJ318427]
97
54
Uncultured Ceratobasidium [EU002954]
Rhizoctonia solani [AJ318433]
Ceratobasidium albasitensis [AJ427398]
52
75
Thanatephorus cucumeris [DQ223780]
Ceratobasidium cornigerum [EU273525]
Ceratobasidium sp. UM01
79
Ceratobasidium sp. [AB286937]
Ceratobasidium sp. [AF354094]
Trametes versicolor [AF042324]
0.1
(a)
58 Fusarium sp. UM78
45 Fusarium oxysporum [HM179532]
Fusarium oxysporum [EF495235]
Fusarium sp. [GQ141219]
Fusarium sp. [EU543261]
Fusarium oxysporum [GU109337]
Fusarium oxysporum UFMGCB 1333 [FJ605243]
Fusarium oxysporum UFMGCB 1376 [FJ605244]
Fusarium oxysporum [GQ365156]
Fusarium sp. UM16
Trametes versicolor [AF042324]
0.1
(b)
88 Scytalidium lignicola [FJ914697]
52 Phoma herbarum [EU715683]
Dothiorella gregaria [AB470835]
Phoma sp. [EF120407]
72
Phoma moricola [GQ352491]
Didymella bryoniae [GU592001]
61
Phyllosticta jasmini [AB470839]
Phoma multirostrata [EF585392]
97
Phoma exigua [EU343173]
Pleosporales sp. [DQ092514]
77 Didymella sp. [DQ092504]
Pleosporales sp. UM29
Trametes versicolor [AF042324]
0.1
(c)
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AJPS
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Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
94 Glomerella cingulata [FJ172237]
44 Colletotrichum gloeosporioides [FJ172224]
95 Fungal endophyte sp. [HM537036]
Colletotrichum gloeosporioides [AJ301979]
Glomerella sp. UM90
Trametes versicolor [AF042324]
0.1
(d)
Colletotrichum sp. [AB443952]
Colletotrichum lineola [GU227843]
Colletotrichum truncatum [AJ301985]
Colletotrichum sublineolum [EU400151]
48 Colletotrichum anthrisci [GU227846]
Colletotrichum fructi [GU227844]
Colletotrichum
sp. [GU227828]
100
69
Colletotrichum dematium [GU227826]
Colletotrichum sp. UM55
Colletotrichum coccodes [AM422216]
Trametes versicolor [AF042324]
0.5
(e)
Mycoleptodiscus indicus UTHSCSA R-4334 [GU220382]
Fungal endophyte sp. ZY-2009 [FJ613801]
Mycoleptodiscus indicus UAMH 8516 [GU980694]
Ascomycota sp. AR-2010 [HQ607907]
Fungal sp. Da1 [HM991177]
Mycoleptodiscus indicus UAMH 8520 [GU980696]
Mycoleptodiscus indicus UAMH 10746 [GU980698]
43 Mycoleptodiscus indicus PA2LL5 [JF736515]
Mycoleptodiscus indicus UM 24
93 Ascomycete sp. Lrub20 [DQ384608]
Ascomycota sp. 3454 [FJ544248]
90
Fungal endophyte sp. AiL8 [EU054412]
Mycoleptodiscus terrestris [EU364807]
Mycoleptodiscus coloratus CBS 720.95 [DQ341499]
Trametes versicolor [AF042324]
0.2
(f)
Figure 1. Sequence analyses were performed based on the rRNA gene sequence (ITS1-5.8S-ITS2) using the maximum composite likelihood model to illustrate the relationship of endophytic fungi phylotypes sequences to closest relatives from GenBank BLAST alignments. The tree was rooted with Trametes versicolor (AF042324) as the outgroup. Letters in bold indicate
sequences obtained in this study, while other sequences represent reference data obtained from GenBank.
Copyright © 2012 SciRes.
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Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
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Table 2. Antifungal activities of crude extracts from endophytic fungi phylotypes isolated from Echinacea purpurea (L.)
Moench. using direct bioautography against three Colletotrichum test species.
Mean Fungal Growth Inhibition of Carvacrol (mm) ± SEMb
Colletotrichum acutatum
a
UM code Endophytic taxa
Colletotrichum fragariae
Colletotrichum gloeosporioides
80 µg/spot
160 µg/spot
80 µg/spot
160 µg/spot
80 µg/spot
160 µg/spot
01
Ceratobasidium sp.
dc
d
d
d
d
d
15
Cladosporium cladosporioides
na
d
d
d
d
d
32
Colletotrichum gloeosporioides
na
na
na
na
na
d
71
C. trifolii
d
d
d
d
na
na
55
Colletotrichum sp.
d
d
d
d
d
d
34
Fusarium oxysporum
4.75 ± 0.8
6.75 ± 2.2
4 ± 1.4
5.5 ± 1.9
2.3 ± 1.3
1.75 ± 1.3
45
F. oxysporum
6.25 ± 1.5
9.25 ± 2.2
6.75 ± 1.41
9 ± 3.46
6.25 ± 0.6
8.75 ± 1.5
68
F. oxysporum
8±0
10 ± 0
5±0
6 ± 1.4
5±0
10.5 ± 0.7
77
F. oxysporum
6±0
9 ± 0.7
6±0
8.5 ± 0.7
4.5 ±0.7
8.5 ± 0.7
57
F. solani
na
na
d
d
d
d
16
Fusarium sp.
7.0 ± 1.4
10.25 ± 2.2
d
d
4.75 ± 0.7
6.25 ± 5.7
58
Fusarium sp.
6±0
10 ± 0
5.5 ± 0.7
9 ± 0.7
5±0
7 ± 0.7
78
Fusarium sp.
6±0
10 ± 0
8±0
10.5 ± 0.7
5.5 ± 0.7
10.5 ± 0.7
24
Mycoleptodiscus indicus
d
d
d
d
7.5 ± 3.56
9 ± 5.74
28
M. indicus
na
na
d
5±0
na
d
29
Pleosporales sp.
na
na
na
d
d
d
d
benomyl
-
d
-
19.5 ± 3.1
-
d
d
captan
-
13.50 ± 1.9
-
14.25 ± 2.2
-
16.25 ± 1.8
d
cyprodinil
-
d
-
25 ± 1.4
-
d
d
azoxystrobin
-
d
-
26 ± 1.4
-
d
a
b
UM = Culture code of Endophytic fungi; Mean inhibitory clear zones and standard errors were used to determine the level of antifungal activity against each
fungal species; cd = Diffuse inhibitory zone. na = no activity. - = no tested; dTechnical grade agrochemical fungicides (without formulation) with different
modes of action were used as internal standards (benomyl at 1.16 µg, captan at 1.2 µg, cyprodinil at 0.9 µg, azoxystrobin at 1.61 µg).
The Fusarium extracts were able to inhibit all Colletotrichum targets. Four F. oxysporum distinct isolates and
two Fusarium sp. (isolates UM16 and UM78) showed a
broad antifungal activities range of 1.75 - 0.5 and 4.75 10.5 µg·spot–1, respectively. Selective fungicidal activity
against C. gloeosporioides was noticed using the crude
extract of phylotype Mycoleptodiscus indicus. This extract also showed a diffuse halo against C. acutatum and
C. fragariae. In contrast, the extracts of phylotypes
Ceratobasidium sp., C. cladosporioides, C. trifolii, Colletotrichum sp., Phomopsis sp., and Pleosporales sp.
showed diffuse zones against the Colletotrichum targets
(Table 2). Diffuse inhibitory zones are regions on the
bioautography plate where fungal growth is visually reduced and interspersed with few mycelia and reproducetive structures and are often characteristic of metabolites
from plant, invertebrate, or microorganisms that are moderate suppressive of fungal growth. Most importantly,
Copyright © 2012 SciRes.
these fungal extracts displayed antifungal activities similar to commercial agrochemical fungicides benomyl, captan, cyprodinil, and azoxystrobin used as control drugs
with halos showing almost the same diameter. Additionally, the extract of phylotype M. indicus UM28 showed
100 and 20% mortality at the concentrations of 125 and
62.5 ppm, respectively, against larvae of A. aegypti.
4. Discussion
The isolates were identified by molecular tools using the
sequencing of the ITS region between the rRNA genes
18S and 26S with sequence length between 495 and 1638
bp. The phylotypes C. cladosporioides, C. gloeosporioides, C. trifolii, F. oxysporum, F. solani, and M. indicus had sequences closely related to the sequences that
are deposited in the GenBank database. According to
Zalar et al. [21], members of the Cladosporium genus are
AJPS
1112
Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
distributed worldwide and C. cladosporioides is one of
the most common endophyte species found in association
with various plant species. The genus Colletotrichum
includes several morphologically similar taxa and can be
found as phytopathogenic, saprophytic, and endophytic
species [22]. Similar findings have been reported for the
species of the genus Fusarium, which was reported as
endophytes symbionts associated with orchids [23] and
angiosperms [5].
An interesting isolate found in E. purpurea was the
phylotype M. indicus, a tropical to subtropical species,
which generally cause necrosis on leaves of many monocotyledonous plants [24]. In addition to be an agent of
leaf necrosis, M. indicus have been described as a human
pathogen causing subcutaneous phaeohyphomycosis [25].
The sequence of UM24 M. indicus was phylogenetically
close to sequences of M. indicus (GU980694, GU980696,
and GU980698) described as agent of subcutaneous
infection in a dog [26]. Although few studies have indicated that endophytic fungi might be quiescent saprobes
or latent pathogens, specific examples detailing these
hypotheses remain scant. To our knowledge, this is the
first work in which M. indicus was isolated as endosymbiont from health leaves of E. purpurea.
Six endophytic phylotypes (UM01, UM16, UM29,
UM55, UM78, and UM90) were analyzed for phylogenetic affinities. The genus Ceratobasidium (Basidiomycota) is cosmopolitan being found as saprotrophic,
pathogenic and as part of an orchid endomycorrhizal
group [27]. Ceratorhiza is the anamorphic genus Ceratobasidium, its species are common endophytes associated with orchids [27]. Glomerella (Ascomycota) is the
anamorphic genus Colletotrichum which includes parasites and saprobes species. Species belonging to Glomerella are distributed worldwide and frequently identified as endophytic fungi [28]. The order Pleosporales is
the largest in the class Dothideomycetes, and according
to Zhang et al. [29] the Pleosporales species can be
endo- or epiphytes as parasites on green leaves or stems,
saprophytic on dead leaves and stems.
Endophytic assemblages were distributed erratically
within the E. purpurea parts. According to Petrini et al.
[30], different plant tissues and organs may represent
distinct microhabitats and Vieira et al. [6] demonstrated
that the composition of endophytic fungal communities
associated with medicinal plants vary within tissues. A
different finding reported by Summerell and Leslie [31]
describes F. oxysporum as pathogenic fungus that infects
the hosts via root system obstructing the vascular system,
thus reducing or preventing the flow of water from the
roots to the upper plant, producing wilting symptoms. In
this study, F. oxysporum was a exception; the fungus was
found asymptomatic within in three different tissues of
Copyright © 2012 SciRes.
healthy E. purpurea and able to produce extracts with
antifungal activity, suggesting a protective symbiosis to
the plant.
Fungal extracts of 16 endophytic fungal isolates were
active against Colletotrichum targets by inhibiting hyphae’s growth and spore germination. Among the isolates are the Cladosporium species, known to produce
several secondary metabolites such as cladosporin, emodin, phytase, taxol and antifouling compounds [32] and
Fusarium species that synthesize several biologically
active metabolites including fusaric acid, moniliformin,
fumonisins, zearalenon, enniatins and trichothecenes [33].
The phylotyes F. oxysporum UM16 and UM78 showed
phylogenetic similarities to two other strains of F. oxysporum (FJ605243, FJ605244), recovered from orchid
species, Epidendrum secundum and Acianthera teres.
Vaz et al. [23] reported that extracts of these F. oxyporum strains had antimicrobial properties against Candida species.
Most of the fungal isolates producing active compounds were associated with plants harvested in their
habitats [23,32,33]. According to Taylor et al. [3], the
removal of species from their natural environment results
in plants more susceptible to attack of different pathogens because disrupt specific or coevolved endophytic
communities. Our results demonstrated differently since
a highly diverse fungal community was removed from
two in vitro propagated clones derived from single E.
purpurea accession (PI631307). The high numbers of
isolates associated with this restricted population indicate
that soil and perhaps organic fertilizer (cotton seed meal)
were the source of inoculums, and healthy plants produced by tissue culture were able to adapt and acquire
their endophytic population. This fungal community presented high dominance index of generalist endophytic
fungi, which were also found in high abundance among
different plant species [34]. The generalist endophytic
fungi in E. purpurea were the phylotypes C. cladosporioides, C. gloeosporioides, and F. oxysporum.
To our knowledge, this is the first report of fungal
community obtained from E. purpurea that include endophytes next to saprobes as well as pathogenic related
species with great potential to produce compounds with
selective antifungal properties, which may be effective in
plant defence and key element for cultivation of high
quality medicinal Echinacea. Most importantly, these
endophytes were isolated from micropropated plants
cultivated in pots and during the process of acclimatization into soil had the community restored.
5. Acknowledgements
This work received partial support of the FAPEMIG
(process CBB 00044/09) and CNPq (process 200774/
AJPS
Diversity and Biological Activities of Endophytic Fungi Associated with Micropropagated Medicinal
Plant Echinacea purpurea (L.) Moench
2011-5). The authors also thank the USDA, Agricultural
Research Service Specific Cooperative Agreement No.
58-6408-7-012 for the research support. The authors
thank Ms. J.L. Robertson, Ms. R. Pace, Ms. X. Wang, Mr.
N. Newlon, Mr. G. Allen, and Mr. W. Reid for performing biological activities. This study was supported by a
grant from the Deployed War-Fighter Protection (DWFP)
Research Program and the US Department of Defense
through the Armed Forces Pest Management Board
(AFPMB).
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