Symbiosis
DOI 10.1007/s13199-015-0316-4
Bioactivity of Azolla aqueous and organic
extracts against bacteria and fungi
Ana L. Pereira & Lucinda J. Bessa & Pedro N. Leão &
Vítor Vasconcelos & P. Martins da Costa
Received: 29 October 2014 / Accepted: 3 February 2015
# Springer Science+Business Media Dordrecht 2015
Abstract Previous reports indicate the use of the aquatic fern
Azolla as medicinal plant in New Zealand and Tanzania
against sore throat and cough, respectively. Therefore, the
aims were to evaluate the bioactivity of Azolla organic and
aqueous extracts against bacteria and yeasts. Organic (dichlo-
romethane:methanol) and aqueous extracts obtained from six
Azolla species were tested against bacterial pathogenic and
non-pathogenic strains (four Gram-positive and three Gram-
negative) and pathogenic fungi (three clinical isolates of
Candida albicans and one of C. glabrata), using the agar
diffusion method and the broth microdilution assay. The re-
sults showed that organic extracts of A. caroliniana and
A. rubra and of A. filiculoides inhibited the growth of
B. subtilis ATCC 6633 whereas those of A. caroliniana and
A. microphylla inhibited the growth of S. aureus ATCC 25923.
The MICs were higher than 4 mg/ml for A. caroliniana,
A. microphylla and A. rubra and higher than 3.25 mg/ml for
A. filiculoides. Aqueous extracts of A. filiculoides,
A. caroliniana, A. microphylla, A. rubra and A. pinnata var.
pinnata induce a small inhibition zone (1 mm) in C. albicans
ATCC 10231 with a MIC higher than 12.5 mg/ml. In conclu-
sion, organic and aqueous extracts of some Azolla species
A. L. Pereira (*) : L. J. Bessa : P. N. Leão : V. Vasconcelos :
P. Martins da Costa
Interdisciplinary Centre of Marine and Environmental Research
(CIIMAR/CIMAR), (Blue Biotechnology and Ecotoxicology),
University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal
e-mail: anapereira271268@yahoo.com
L. J. Bessa : P. Martins da Costa
ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Rua de
Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
V. Vasconcelos
Department of Biology, Faculty of Sciences of the University of
Porto, Rua do Campo Alegre, 4069-007 Porto, Portugal
show potential for use against infections caused by Gram-
positive bacteria and C. albicans, respectively.
Keywords Azolla . Antifungal . Antimicrobial . Bioactivity .
Extracts
1 Introduction
Azolla spp. are small free-floating aquatic pteridophytes, be-
longing to the family Azollaceae, with a worldwide distribu-
tion on tropical, subtropical and temperate regions. This fern is
unique as it has an everlasting symbiosis with the nitrogen-
fixing cyanobacterium Anabaena azollae inhabiting cavities
in the Azolla dorsal lobes. This fern, although considered an
invasive species, can be used as biofertilizer, in the
phytoremediation of domestic and industrial wastewater and
as animal or human food (Wagner 1997; Carrapiço 2010).
Despite the scarce ethnopharmacological data on the sym-
biosis Azolla-A. azollae, it has been indicated as having me-
dicinal potential. In the 16th century, the Chinese Li Shi-zhen
(cited by Shi and Hall 1988) described Azolla as having me-
dicinal properties but with no specification to which disease.
In addition, Usher (1974) pointed the use of Azolla in New
Zealand to cure sore throat when chewed and Wagner (1996)
indicated this fern as a traditional cough medicine in Tanzania.
Given that this symbiosis exists worldwide, local human pop-
ulations may use it for their benefit. However, the potential use
of Azolla in medicine or pharmacology and possible patho-
genic targets remains not explored due to a lack of bioactivity
data. One of the few reports about bioactivity was performed
with the extracts from the cyanobiont of A. filiculoides in
which the lipophilic extract had activity against Penicillium
expansum (a fungus that is a plant pathogen), while the
 Pereira A.L. et al.

hydrophilic extract presented activity against Agrobacterium
vitis (a bacterial plant pathogen) and against the crustacean
Artemia salina (Piccardi et al. 2000). More recently, the meth-
anolic extract of A. microphylla showed antimicrobial activity
against Xanthomonas spp., also a plant pathogen (Abraham
2013).
The aim of the present research was to evaluate the
biological activities of organic and aqueous extracts
from six Azolla species against bacteria and yeasts with
clinical relevance.
temperature. The supernatant was frozen, lyophilized until
dryness, weighted and stored at −80 °C. Prior to the bioassays,
all extracts were resuspended in DMSO to obtain a stock
concentration of 100 mg/ml, except for the organic extract
of A. filiculoides, with a stock concentration of 81.58 mg/ml
and stored at −20 °C.
2.3 Antimicrobial activity
2.3.1 Bacterial and yeast strains

The organic and aqueous extracts were tested against four

2 Material and methods
2.1 Plant material
Six Azolla species from the germplasm collection at IRRI
(International Rice Research Institute) (Table 1) were cultured
in Hoagland medium (H-40), pH 6.1–6.2, with controlled
temperature, photoperiod and light intensity (Pereira and
Carrapiço 2009). The collected biomass was washed in water,
stored at −80 °C, lyophilized and weighted.
2.2 Extraction of aqueous and organic compounds
The lyophilized biomass of Azolla was ground in a mill
and extracted twice with dichloromethane:methanol (2:1,
v/v) for 3 h, vacuum filtered through paper filter
(Whatmann N° 1) and cheese cloth and evaporated until
dryness in a rotary evaporator (Büchi, Maia, Portugal)
thus yielding the organic fraction which was weighted
and stored at −80 °C.
To obtain the aqueous fraction, the organic solvent-
extracted biomass was extracted twice with MiliQ ultrapure
water for 2 h, sonicated with an ultrasound probe at 20 kHz for
7 min., filtered through a cheese cloth and paper filter
(Whatmann N° 1), centrifuged at 4542 g for 30 min. at room
Gram-positive bacteria (Bacillus subtilis ATCC 6633,
Staphylococcus aureus ATCC 25923, Enterococcus faecalis
ATCC 29212, Staphylococcus aureus B1 (MRSA, exhibiting
the characteristics of the pandemic EMRSA-15, isolated from
public buses)), three Gram-negative bacteria (Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Acinetobacter baumannii A2 (a multidrug-resistant clinical
isolate) and four strains of Candida spp. (Candida albicans
ATCC 10231, C. albicans isolated from faeces (CA190349),
C. albicans isolated from a vaginal exudate (CA190464) and
C. glabrata isolated from a vaginal exudate (CG188901)).
2.3.2 Agar-well diffusion method
Overnight fresh grown bacterial colonies were used to prepare
the bacterial inocula in Mueller-Hinton broth (MHB) (BioKar
Diagnostics, Beauvais, France) equal to 0.5 McFarland. Ten-
fold serial dilutions were performed and the third obtained
dilution was incorporated in Mueller-Hinton agar (MH)
(BioKar Diagnostics) previously cooled to 45 °C, which was
then poured into a Petri dish. After agar solidification, wells
(6 mm in diameter) were punched in the agar using a sterile
glass tube. The wells were loaded with 50 μl of the each
extract; plates were let at room temperature for 2 h to allow
the extract diffusion before incubation at 37 °C for 24 h. The

Table 1 Origin of the Azolla species used to extract compounds for the bioassays

Azolla species and IRRI codea Origin and harvest year Sourceb or collector

A. pinnata var. imbricata, PI1 Philippines, Sto Domingo, Albay, 1975 IRRI
A. filiculoides, FI1001 East Germany (ex.- GDR), 1979 IB China
A. mexicana, ME2026 Brazil, Solimoes river, Pacencia Island, Iranduba, Amazonas T. Lumpkin
(BLCC 18), 1984
A. caroliniana, CA3525 Ruanda, Cyili Rice Research Center, 1987 C. van Hove
A. microphylla, MI4021 Equator, Santa Cruz Island, Galapagos, 1982 T. Lumpkin
A. rubra, RU6010 New Zealand, Nouville, 1986 C. van Hove
A. pinnata var. pinnata, PP7509 Nigeria, Moor plantation, 1987 C. van Hove

a
The accession numbers were listed according to the IRRI code
b
IB China-Institute of Botany, Academia Sinica, Beijing, China, IRRI-International Rice Research Institute
Bioactivity of Azolla aqueous and organic extracts against
DMSO was used as a solvent control. The antibacterial activ-
ity of each extract was evaluated by measuring the inhibition
zone (mm) around the well.
2.3.3 Agar-disc diffusion method
The antifungal activity of the Azolla extracts was performed
using the disc diffusion method. Fresh yeast colonies grown in
Sabouraud agar (BioKar Diagnostics) were inoculated in liq-
uid RPMI-1640 medium (BioWhittaker, Lonza, Walkersville,
USA) to obtain fungi suspensions of 0.5 McFarland. The yeast
inocula were spread into Sabouraud agar plates. Blank discs
with 6 mm in diameter placed on the surface of the inoculated
plate were impregnated with 15 μl of the stock of each extract.
The DMSO was used as a control. Plates were kept for 2 h at
room temperature to allow the extract diffusion and then in-
cubated at 37 °C for 18–24 h. Inhibition zones around the
discs were compared with the control (DMSO) and measured
(mm).
2.3.4 Microdilution method – MIC determination
To determine the minimum inhibitory concentration (MIC) of
Azolla extracts against both bacterial or yeast strains, the broth
microdilution method was used according to the recommen-
dations of the Clinical and Laboratory Standards Institute
(CLSI 2012). Extract solutions were serial diluted in MHB
(for bacterial strains) or in RPMI-1640 (for yeast) to
achieve in-test concentrations ranging from 0.125 to
4 mg/ml (in the case of the A. filiculoides organic ex-
tract was 0.10 to 3.25 mg/ml) or 0.19 to 12.50 mg/ml,
respectively. Bacterial inocula were prepared in MHB
and yeast inocula in RPMI-160 medium and standard-
ized in order obtain a concentration of 5 × 105 CFU/ml
in each inoculated well of the microtiter plate. The
DMSO was used as a control. The MIC was defined
as the lowest concentration of extract that had inhibited
the growth, evaluated by measuring the absorbance at
595 nm before and after the incubation using an iMark
Microplate Reader (BioRad, Hércules, California, USA).
Fig. 1 Antimicrobial activity of
organic extracts of Azolla against
B. subtilis ATCC 6633. The
inhibition zone (mm) was
measured around the wells. a
A. filiculoides (FI 1001); b
A. caroliniana (CA 3525); c
A. rubra (RU 6010).
3 Results and Discussion
In the three reports regarding the use of Azolla as a medicinal
plant, the species used were probably different. In China, it is
possible to find A. rubra, A. caroliniana and A. pinnata var.
imbricata; New Zealand has A. rubra and A. pinnata var.
imbricata; and Tanzania has A. nilotica and A. pinnata var.
pinnata (Lumpkin and Plucknett 1980; Wagner 1997; Pereira
et al. 2011). The present research is the first report
about the bioactivity of organic and aqueous extracts
of six Azolla species against clinical important bacterial
and yeast strains.
The organic extracts showed no bioactivity against
Candida spp. and Gram-negative bacteria, whereas the aque-
ous extracts had no activity against any of the Gram-positive
and Gram-negative bacteria tested. Regarding the Gram-
positive bacteria, the organic extracts from A. filiculoides
(Fig. 1a), A. caroliniana (Fig. 1b) and A. rubra (Fig. 1c)
inhibited the growth of B. subtilis ATCC 6633 with an inhibi-
tion zone around the well of 3, 1.2 and 1 mm, respectively.
The growth of S. aureus ATCC 25923 was inhibited by the
organic extracts of A. caroliniana (Fig. 2a) and A. microphylla
(Fig. 2b) with an inhibition zone of 1.2 and 1.1 mm, respec-
tively. The MIC was higher than 4 mg/ml for the organic
extracts of A. caroliniana and A. rubra and 3.25 mg/ml for
the A. filiculoides organic extract. The reports about the me-
dicinal use of Azolla indicate their use in New Zealand to cure
sore throat (Usher 1974), an inflammation in the oropharynx
cavity caused by the Gram-positive bacterium S. pyogenes or
by trauma. Although it was not possible to test the extracts
against S. pyogenes, the present research used the Gram-
positive bacterium S. aureus that can cause respiratory, skin
and nosocomial infections. The organic extract of
A. filiculoides, a species native in New Zealand (Lumpkin
and Plucknett 1980), inhibited the growth of this bacterium
that reinforces the report of their traditional medicinal use.
However, the extract of A. pinnata var. pinnata, a species
native in Tanzania and reported to be use for cough (Wagner
1996) did not inhibit the growth of S. aureus, one of the
bacteria that can cause cough. In addition, this research reports
for the first time the ability of Azolla to inhibit the growth of
B. subtilis, a gastrointestinal and soil pathogen. It may thus be
 Pereira A.L. et al.

Fig. 2 Antimicrobial activity of organic extracts of Azolla against

S. aureus ATCC 25923. The inhibition zone (mm) was measured
around the wells. a A. caroliniana (CA 3525); b A. microphylla (MI
4021).

possible to use this fern to control intestinal diseases caused by

this bacteria. But another ferns belonging to the Salviniaceae
family and phylogenetically related to Azolla, such as Salvinia
auriculata (Lima et al. 2013) and S. minima (Panda et al.
2014) have been shown to inhibit the growth of S. aureus
while S. molesta inhibited B. cereus (Mandal and Mondal
2011).
The antifungal potential of Azolla extracts was assessed
against yeasts. The aqueous extracts did not showed bioactiv-
ity against C. albicans (CA190349 and CA190464) or
C. glabrata. However, the aqueous extracts from
A. filiculoides (Fig. 3a), A. caroliniana (Fig. 3b),
A. microphylla (Fig. 3c), A. rubra (Fig. 3c) and A. pinnata Fig. 3 Antifungal activity of aquatic extracts of Azolla against
var. pinnata (Fig. 3d), were mildly bioactive against C. albicans ATCC 10231. The inhibition zone (mm) was measured
around the discs. a A. filiculoides (FI 1001); b A. caroliniana (CA
C. albicans ATCC 10231 with an inhibition zone of 1 mm 3525); c A. microphylla (MI 4021); d A. rubra (RU 6010); e A. pinnata
and a MIC above 12.5 mg/ml. To our knowledge, there are no var. pinnata (PP 7509).
reports stating antifungal activity for Azolla aqueous extracts.
The yeast C. albicans although being a commensal of the activities may be present in very low levels in the pool of
gastrointestinal tract, can cause oral and vaginal diseases in compounds that compose the extract.
humans. The only report using extracts from the cyanobiont of
A. filiculoides showed inhibition in P. expansum, a plant fun-
gus (Piccardi et al. 2000). But, the extracts of another fern, 4 Conclusions
S. minima, inhibited the growth of C. albicans (Panda et al.
2014). The data from the present study has showed that the organic
Although the present research showed extracts from three and two Azolla species were promising in
ethnopharmacologically relevant bioactivities, the Azolla’s the growth inhibition of B. subtilis ATCC 6633 and S. aureus
metabolites that can cause the inhibition of Gram-positive ATCC 25923, respectively. In addition, the aqueous extracts
bacteria and fungi were not purified nor identified and the of A. filiculoides, A. caroliniana, A. microphylla, A. rubra and
analysis of Azolla metabolites should be further explored. A. pinnata var. pinnata inhibited the growth of the yeast
Even so, some compounds already identified in Azolla species C. albicans. This means that the two reports concerning their
could be related with the activities demonstrated in this re- medicinal use could be accurate and the people that have
search. The phenylpropanoids α-asarone and isoeugenol access to Azolla that grows in rivers, dams or creeks may
methyl ether of A. filiculoides (Greca et al. 1989), the volatile potentially benefit by its usage on some Gram-positive bacte-
compounds such trans-2-hexenal and geraniol of rial infections. Still, more research is necessary to screen other
A. filiculoides (Pereira et al. 2009), the anthraquinones of bacteria and fungi strains, but also to isolate and identify the
A. microphylla (Abraham and Aeri 2012) and the flavonoids, compound or compounds responsible of those bioactivities.
phenols or coumarins of A. microphylla (Abraham 2013) can
have bioactivity against fungi and bacteria. The high MIC
Acknowledgments This research was partially supported by 1) Euro-
values found for both antibacterial and antifungal extracts pean Regional Development Fund (ERDF) through the COMPETE (Op-
probably indicate that compounds responsible for those erational Competitiveness Programme) and national funds through FCT
Bioactivity of Azolla aqueous and organic extracts against
(Foundation for Science and Technology)- under the project PEst-C/
MAR/LA0015/2013; and 2) the Project MARBIOTECH (reference
NORTE-07–0124-FEDER-000047), co-financed by the North Portugal
Regional Operational Programme (ON.2 – O Novo Norte), under the
National Strategic Reference Framework (NSRF), through the European
Regional Development Fund (ERDF). The European Social Funding
(FSE) under the Human Potential Operational Program (POPH) of Na-
tional Strategic Reference Board (QREN) supports the fellowship
SFRH/BPD/44459/2008 to Ana L. Pereira. Thanks to Stephan Haefele
and Agnes Padre of IRRI for sending Azolla and Cristina Veríssimo
(Laboratory of Mycology, Instituto Nacional de Saúde Dr. Ricardo Jorge,
Lisboa, Portugal) for providing the yeasts strains.
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