Pharmaceutical Biology

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Screening of Soil Cyanobacteria for Antifungal and

Antibacterial Activity

N. Soltani, R.A. Khavari-Nejad, M. Tabatabaei Yazdi, Sh. Shokravi & E.

Fernández-Valiente

To cite this article: N. Soltani, R.A. Khavari-Nejad, M. Tabatabaei Yazdi, Sh. Shokravi & E.
Fernández-Valiente (2005) Screening of Soil Cyanobacteria for Antifungal and Antibacterial Activity,
Pharmaceutical Biology, 43:5, 455-459, DOI: 10.1080/13880200590963871

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Pharmaceutical Biology
2005, Vol. 43, No. 5, pp. 455–459

Screening of Soil Cyanobacteria for Antifungal

and Antibacterial Activity

N. Soltani1, R.A. Khavari-Nejad2, M. Tabatabaei Yazdi3, Sh. Shokravi4, and E. Fernandez-Valiente5

1
Department of Biology, ACECR, Research Institute of Applied Science, University Shahid Beheshti, Tehran, Iran;
2
Department of Biology, Faculty of Science, Teacher Training University, Tehran, Iran; 3Department of Pharmaceutical
Biotechnology, Faculty of Pharmacy, Tehran University of Medical Science, Tehran, Iran; 4Department of Biology,
Faculty of Science, Islamic Azad University, Unit Gorgan, Gorgan, Iran; 5Department of Biology, Faculty of Science,
University Autonoma de Madrid, Madrid, Spain

Abstract
Soil cyanobacteria isolated from the paddy fields of
seven provinces in Iran was evaluated for antimicrobial
activity. Aqueous, petroleum ether, and methanol
extracts from 76 microalgae were examined for anti-
microbial properties against four bacteria and two fungi.
Of total microalgae, 22.4% (17 cyanobacteria) exhibited
antimicrobial effects. Selected cyanobacteria with posi-
tive antimicrobial activities were members of the families
Stigonemataceae, Nostocaceae, Oscillatoriaceae, and
Chroococcaceae. Growth of Bacillus subtilis Persian
Type Culture Collection (PTCC) 1204 and Staphylo-
coccus epidermidis PTCC 1114 were inhibited by 12 and
14 species of cyanobacteria, respectively. Also, eight cya-
nobacteria inhibited the growth of Escherichia coli
PTCC 1047, and two species inhibited the growth of
Salmonella typhi PTCC 1108. Considering fungi, six
species inhibited the growth of Candida kefyr ATCC
1140, and one cyanobacterium species inhibited the
growth of Candida albicans ATCC 14053. Furthermore,
one of the paddy fields (Khozestan) showed no anti-
microbial activity.
Keywords: Antibiotics, antimicrobial activity, cyano-
bacteria, paddy fields, soil.
Introduction
Cyanoacteria are a major component of microbial flora
in rice paddy fields and contribute to the fertility of such
ecosystems (Roger & Kulasooriya, 1980). Producing
active biocide components could be another important
selective advantage (Mundt et al., 2001). Terresterial
and marine cyanobacteria have proved to be an
extremely valuable source of novel bioactive agents
(Jaki et al., 2000; Hirata et al., 2000).
Screening of cyanobacteria for antibiotics and other
pharmacologically active compounds has recently
received considerable attention, cyanobacteria are as
novel bioactive natural producers (Borowitzka, 1995;
Borowitzka & Borowitzka, 1988). Many active sub-
stances with antibacterial, antiviral, fungicide, enzyme
inhibiting, immunosuppressive, cytotoxic, and algicide
activity have been isolated from cyanobacterial biomass
or in some cases from the medium of laboratory cultures
(Knubel et al., 1990; Mule et al., 1991; Gerwork et al.,
1994; Falch et al., 1995; Jaki et al., 1999).
Cyanobacteria of local habitats have rarely been
screened for biological effects. Ghasemi et al. (2003) have
surveyed antibacterial activities of microalgae of northern
Iran. Also, a novel antimicrobial substance named
Parsiguine has been identified (Ghasemi et al., 2004).
Therefore, in a continuing investigation, the biological
activities of various species and strains of terrestrial cya-
nobacteria in different provinces of Iran were studied.
The ability to produce antimicrobial substances may be
noticed not only as a defensive instrument for the strains
but also as a good source of new bioactive compounds
from a pharmaceutical point-of-view. Accordingly, 228
lipophilic and hydrophilic extracts obtained from 76
samples of cultured soil cyanobacteria were investigated
to detect their antimicrobial effects.

Accepted: March 21, 2005

Address correspondence to: Neda Soltani, Department of Biology, ACECR, Research Institute of Applied Science, University Shahid
Beheshti, P.O. Box 19835-371, Tehran, Iran. Tel.: 0098 21 2402097; Fax: 0098 21 2418799; E-mail: soltani6@yahoo.com

DOI: 10.1080/13880200590963871 # 2005 Taylor & Francis Ltd.

456 N. Soltani et al.

Materials and Methods standard (antimicrobial index):

Materials Inhibition zone of sample  100
Inhibition zone of the standard
All of the chemicals and reagents used in the current study
were purchased from Merck (Darmstadt, Germany).
Gentamicin and nystatin were obtained from Sigma Results
(St. Louis, MO, USA).
Soil cyanobacteria for this study were isolated from
different geographical locations, mainly north of Iran
Methods due to high distribution of paddy fields in these regions.
Isolation and culture conditions Specification of species and their original habitats are
listed in Table 1.
Soil algae were isolated from paddy fields of different Seventy-six strains were isolated. All of them were
provinces in Iran: Golestan, Mazandaran, and Gillan cultured in an inorganic medium (BG110) at 30C with
(north); Isfahan and Fars (central); Ilam (west) and illumination. The harvested cells were successively
Khoozestan (south). Soil samples were cultured by usual extracted with solvents of increasing polarity (petroleum
methods (Kaushik, 1987). Algae were grown in 250-ml ether and methanol, respectively). The supernatants were
conical flasks containing 100 ml of BG11 medium extracted separately. Antimicrobial activity was shown
adjusted to pH 7.4. The cultures were illuminated con- by 17 isolated species. Five species belonged to the genus
tinuously (50 mE m
2 s
1) supplied by six fluorescent Nostoc. Nine of them were included in the family Stigo-
lamps and following incubation at 30  1C. Identifi- nemataceae (three Stigonema, four Hapalosiphon, two
cation of cyanobacteria with antimicrobial activity was Fischerella). Others belonged to the genera Plectonema,
done according to Desikachary (1959), Prescott (1962), Oscillatoria, and Chroococcus (Table 2).
Anagnostidis & Komarek (1990), and John et al. (2003). Between the fungi, Candida albicans ATCC 14053 was
more resistant. It was inhibited by only one species. The
cell extracts and culture medium of 10 strains such as
Antimicrobial bioassay Oscillatoria, Hapalosiphon, Fischerella, and Stigonema
Cells were harvested by centrifugation at 5000  g for species showed antifungal activities against C. kefyr
15 min, followed by extraction with petroleum ether ATCC 38296. But among all strains, only the petroleum
and methanol. The aqueous supernatants and solvent ether and methanol extracts of H. hibernicus West &
extracts were concentrated under reduced pressure at West FS 33 showed antifungal activity against C. albi-
40C. The antibacterial and fungicide activities of algal cans ATCC 14053. The same results were achieved in this
extracts were evaluated by agar plate diffusion test cyanobacterium in relation to C. kefyr ATCC 38296
(Lorain, 1996). (Table 3).
The following bacteria and fungi were used as test
organisms: Gram-positive bacteria, Staphylococcus epi-
dermidis (Winslow & Winslow) Schleifer & Kloos PTCC Table 1. Investigated species with antimicrobial activity and
1114 and Bacillus subtilis (Ehrenberg) Cohn PTCC 1204; their geographical origin.
Gram-negative bacteria, Escherichia coli (Migula) Castel- No. Strain Species Origin
lani & Chalmers PTCC 1047 and Salmonella typhi
(Schroeter) Warren & Scott PTCC 1108; Fungi, Candida 1 FS 23 Stigonema ocellatum Golestan
kefyr (Beij.) Uden & H. R. Buckley ex S. A. Mey. & 2 FS 30 S. ocellatum Mazandaran
3 FS 14 S. turfaceum Isfahan
Ahearn ATCC 38296, and Candida albicans (C. P.
4 FS 21 Nostoc microscopicum Ilam
Robin) Berkhout ATCC 14053.
5 FS 13 N. muscorum Golestan
Filter-paper disks (6.4 mm) were saturated with 60 ml 6 FS 4 N. sp. Golestan
of the test solution, dried, and placed on Müller-Hinton 7 FS 5 N. punctiforme Mazandaran
agar plates for bacteria and Saubouraud’s dextrose agar 8 FS 8 N. linckia Mazandaran
plates for fungi inoculated with a lawn of the test micro- 9 FS 6 Hapalosiphon brasiliensis Gillan
organisms. 10 FS 3 H. aureus Gillan
Plates were incubated at an appropriate temperature 11 FS 7 H. fontinalis Fars
for bacteria (37C) and fungi (25C) for a period of 12 FS 33 H. hibernicus Fars
18–24 h in dark. Gentamicin and nystatin disks (10 mg) 13 FS 2 Fischerella ambigua Fars
were used as positive controls. The distinct and circular 14 FS 18 F. ambigua Gillan
15 FS 28 Plectonema notatum Golestan
radii of inhibition zones were measured.
16 FS 19 Oscillatoria angusta Mazandaran
The following formula was used for comparison of the
17 FS 1 Chroococcus sp. Mazandaran
antimicrobial activity of the sample with that of the
 Antimicrobial activity of cyanobacteria
Table 2. List of cyanobacteria with antimicrobial activity.
Order Family Genera
Chroococcales Chroococcaceae Chroococcus sp.
Oscillatoriales Oscillatoriaceae Oscillatoria sp.
Plectonema sp.
Nostocales Nostocaceae Nostoc spp.
Stigonematales Stigonemataceae Stigonema spp.
Fischerella spp.
Hapalosiphon spp.
Also, H. hibernicus FS 33 indicated a widespread
spectrum of antimicrobial activities. Only two cyano-
bacteria indicated antibacterial activity against Salmon-
ella typhi PTCC 1108, and these results were detected
in petroleum ether extracts.
Results showed that in species with activity against
S. epidermidis PTCC 1114, the Ai (Antimicrobial index)
varied between 32 and 112. This is comparable with the
results of Ghasemi et al. (2003). It was different in other
cases, for instance 38–78 against B. subtilis PTCC 1204
and 32–86 against E. coli PTCC 1047. With respect to
S. typhi PTCC 1108, the activity index was around 84
and changed to 83–175 in experiments with C. kefyr
ATCC 38296. But it decreased to 80 in relation to C. albi-
cans ATCC 14053. On the other hand, Fischerella ambi-
gua (Naeg.) Gomont FS18 has the highest Ai (112) in the
methanol extract against S. epidermidis PTCC 1114. It
seems this species may have potent antimicrobial activity
and can be the subject of future experiments.
Discussion
In the past decades, screening programs have revealed
that cyanobacteria are a potential source of new active
substances for medicine and pharmacy, and numerous
active compounds have been isolated (Moore et al.,
1989; Falch et al., 1995; Jaki et al., 1999). But few have
been done with respect to cyanobacteria from paddy
fields. Possibly, the synthesis of highly active toxin can
be a defense option of cyanobacteria in these environ-
ments against other organisms like bacteria, fungi,
viruses, and eukaryotic microalgae (Mundt et al., 2001).
Cyanobacteria from the paddy fields of northern
Thailand have produced bioactive substances with anti-
biotic activity against B. subtilis (Chetsumon et al.,
1993). This is in agreement with results obtained in the
current project in which cell extracts and culture media
of 11 species had activity against B. subtilis.
Also, extracts of terrestrial Fischerella ambigua were
the most active in the test systems used (Falch et al.,
1995). The polychlorinated aromatics, ambigols A and
B, were isolated as the agents responsible for the pro-
nounced antimicrobial activities of the lipophilic
extracts. These activities were also well established in
457
the current observations. Petroleum ether, methanol,
and supernatant extracts of F. ambigua FS18 had inhibit-
ony activity against B. subtilis PTCC 1204, S. epidermidis
PTCC 1114, and E. coli PTCC 1047. In addition, cell
extracts of this strain had antifungal activity against
C. kefyr ATCC 38296.
As mentioned, few studies have been done in relation
to cyanobacteria of Iran (Ghasemi et al., 2003), and this
study follows a screening program for isolation and
identification of antimicrobial-producing species from
Iranian paddy fields. No inhibition was shown against
E. coli PTCC 1047 and no antimicrobial activity was
detected in nonpolar extracts (Ghasemi et al., 2003). In
spite of that, data obtained from this research demon-
strated antimicrobial activity against E. coli PTCC
1047 in six methanol, three petroleum ether, and one
aqueous extract. A diterpenoid with antibacterial activity
from Nostoc commune Vaucher against E. coli who iso-
lated by Jaki et al. (1999).
No inhibition was observed against C. albicans ATCC
14053 by a methanol extract of Nostoc muscorum C. A.
Agardh FS 13. This disagreed with the observations of
Cano et al. (1990), who have evaluated the antifungal
activity of terrestrial cyanobacterium N. muscorum
against the same fungus by 20.83%. Mule et al. (1991)
also indicated that phenolic compounds of N. muscorum
exert an inhibitory effect on C. albicans.
The antibacterial effect against S. typhi was only
found in more lipophilic extracts (petroleum ether).
The aqueous extracts were ineffective, a phenomenon
that has already been described by Mundt et al. (2001).
The antimicrobial activities of algal extracts are shown
in Tables 1 and 3. Data in Table 3 show the cyanobacteria
with antimicrobial effects belong to the following families.
Stigonemataceae: Stigonema ocellatum (Dillw.) Thuret FS
23, S. turfaceum (Berkeley) Cooke FS 14, S. ocellatum FS
30, H. brasiliensis Borge FS 6, H. aureus West & West
FS 3, H. fontinalis (Agardh) Bornet FS 7, H. hiberinicus
FS 33, Fischerella ambigua FS 2, and F. ambigua FS 18;
Nostocaceae: Nostoc muscorum C. A. Agardh FS 13,
Nostoc sp. FS 4, N. linckia (Roth) Bornet & Thuret FS 8,
N. microscopicum Carmichael FS 21, and N. punctiforme
C. A. Agardh FS 5; Oscillatoriaceae: Plectonema notatum
Schmidle FS 28 and Oscillatoria angusta Koppe FS 19;
Chroococcaceae:Chroococcus sp. FS 1.
Apparently, the potent antibacterial cyanobacteria
from different localities of Iran belong to Stigonemata-
ceae and Nostocaceae. These results are in agreement
with other experiments in which cyanobacteria such
as H. fontinalis (Moore et al., 1987), F. ambigua (Falch
et al., 1995), F. musciola (Borzi) Gomont (Hagmann &
Jüttner, 1996), N. spongiaeforme C. A. Agardh (Hirata
et al., 2000), and N. commune (Jaki et al., 2000) produce
antimicrobial substances.
In a few cases, species like Plectonema notatum FS 28
(Golestan Province) and Oscillatoria angusta FS 19
458 N. Soltani et al.

Table 3. Antimicrobial activity of different extracts from cyanobacteria.

Bioautographic assay

B. subtilis S. epidermidis E. coli S. typhi C. kefyr

Strain Extract PTCC 1204 PTCC 1114 PTCC 1047 PTCC 1108 ATCC 38296 C. albicans ATCC 14053

FS 23 MeOH þ þ þ
þ

FS 30 H2O
þ




FS 14 PE
þ

þ

H2O þ þ




FS 21 PE þ





MeOH þ þ þ



FS 13 MeOH þ þ þ



FS 4 PE þ





FS 5 PE þ

þ


FS 8 PE

þ



H2O
þ




FS 18 PE þ þ þ
þ

MeOH þ þ þ
þ

H2O þ þ þ



FS 6 H2O
þ




FS 3 PE



þ

H2O þ þ




FS 7 MeOH þ þ þ



H2O
þ




FS 33 PE þ þ þ þ þ þ
MeOH þ þ

þ þ
H2O þ þ

þ

FS 2 MeOH
þ þ



FS 28 PE þ





FS 19 PE þ


þ

MeOH þ þ




H2O
þ

þ

FS 1 MeOH
þ

PE, petroleum ether extract; MeOH, methanol extract; H2O, aqueous extract.

(Mazandaran Province) showed antibacterial effects
against B. subtilis PTCC 1204 and S. epidermidis PTCC
1114. Antimicrobial activity of Chroococcus sp. FS 1
was observed in one paddy field in Mazandaran Province
against S. epidermidis PTCC 1114 (Tables 1 and 3).
A variety of solvents with different polarities were used
for the extraction of algal bioactive materials. All extracts
showed antimicrobial activity, probably because of the
different polarity of active components. But the percentage
of methanol extracts with positive effects was higher, indi-
cating a higher chance of finding antimicrobial activity in
methanol extracts. It was found that active compounds
was higher against Gram-positive bacteria (S. epidermidis
PTCC 1114, B. subtilis PTCC 1204) in comparison with
Gram-negative bacteria (E. coli PTCC 1047, S. typhi PTCC
1108), the same results that Ghasemi et al. (2003) have
observed. Gram-positive bacteria (S. epidermidis PTCC
1114, S. aureus Rosenbach PTCC 1112, S. haemolyticus
Schleifer & Kloos ATCC 29970) are more sensitive than
Gram-negative bacteria [Shigella sonnei (Levine) Weldin
PTCC 1325, Escherichia coli PTCC 1047, and Proteus
vulgaris Hauser PTCC 1312].
Seventeen species from different localities showed
antimicrobial effects. The extracts were most effective
against S. epidermidis PTCC 1114 and to a lesser extent
S. typhi PTCC 1108. Ten extracts inhibited E. coli PTCC
1047. Most inhibition was produced by methanol
extracts. These results disagreed with Ghasemi et al.
(2003) and Kellam and Walker (1989).
The most potent species, Hapalosiphon hiberinicus FS
33, was collected from different paddy fields of Fars
Province. Interestingly, specimens of this species from
paddy fields of Khoozestan Province showed no antibac-
terial production. The cyanobacteria of paddy fields of
Iran showed different antimicrobial behaviors (Tables 1
and 3).
It was interesting that some species showed similar
characteristics. For example, F. ambigua FS18 that was
collected from Gillan Province showed very strong and
permanent antibacterial abilities, agreeing with reported
activity by Ghasemi et al. (2003) for the same species
collected in Golestan Province.
It seems that among all of the species studied in the
current work for antibacterial and antifungal activity,
 Antimicrobial activity of cyanobacteria
Fischerella and Hapalosiphon had most prominent effects
and display a potential that warrants further investigation.
Data also indicated that the highest percentage of colo-
nization and distribution of cyanobacteria in soil cultures
and also the highest rate of antimicrobial activity belonged
to northern provinces (70.5% in Golestan, Gillan, and
Mazandaran Provinces). The lower colonization was
seen in Fars (17.7%), Isfahan (5.9%), and Ilam (5.9%),
respectively.
As results indicated (Table 1), none of isolated sam-
ples from Khoozestan Province (south of Iran) demon-
strated antimicrobial activity. Also, the distribution and
colonization of microalgae in these cultures were lower
than other locations. Nostoc microscopicum, which was
the dominant species in these paddy fields, exhibited no
antimicrobial ability (data not shown). The current
results show that most of the investigated cyanobacteria
induced a response in at least one of the test systems
applied. The most promising cyanobacterial strains for
further studies appeared to be Fischerella ambigua FS
18, Hapalosiphon hibernicus FS 33, and Oscillatoria angu-
sta FS 19.
Acknowledgments
The authors wish to thank Dr. Y. Ghasemi and Mr. G.
Zarrini for collection and spectral studies. Mrs. Ladan
Baftehchi is also thanked for her participation in isolation
of microalgae.
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