30 - medicinal plants
TRANSCRIPT
ORIGINAL PAPER
Actinomycetes isolated from medicinal plant rhizosphere soils:diversity and screening of antifungal compounds, indole-3-aceticacid and siderophore production
Sutthinan Khamna Æ Akira Yokota ÆSaisamorn Lumyong
Received: 24 July 2008 / Accepted: 27 November 2008 / Published online: 16 December 2008
� Springer Science+Business Media B.V. 2008
Abstract A total of 445 actinomycete isolates were
obtained from 16 medicinal plant rhizosphere soils. Mor-
phological and chemotaxonomic studies indicated that 89%
of the isolates belonged to the genus Streptomyces, 11%
were non-Streptomycetes: Actinomadura sp., Microbispora
sp., Micromonospora sp., Nocardia sp, Nonomurea sp. and
three isolates were unclassified. The highest number and
diversity of actinomycetes were isolated from Curcuma
mangga rhizosphere soil. Twenty-three Streptomyces iso-
lates showed activity against at least one of the five
phytopathogenic fungi: Alternaria brassicicola, Collecto-
trichum gloeosporioides, Fusarium oxysporum, Penicillium
digitatum and Sclerotium rolfsii. Thirty-six actinomycete
isolates showed abilities to produce indole-3-acetic acid
(IAA) and 75 isolates produced siderophores on chrome
azurol S (CAS) agar. Streptomyces CMU-PA101 and
Streptomyces CMU-SK126 had high ability to produced
antifungal compounds, IAA and siderophores.
Keywords Actinomycetes � Antagonistic �Indole-3-acetic acid � Siderophores � Biocontrol
Introduction
Microorganisms have been shown to be attractive sources
of natural compounds for the pharmaceutical and other
industries. In agriculture, phytopathogenic fungi can cause
plant diseases and much loss of crop yields. Pesticides are
used to control plant diseases. However, agrochemical
treatment causes environmental pollution and decreased
diversity of non-target organisms. Microorganisms as bio-
logical control agents have high potential to control plant
pathogens and no effect on the environment or other non-
target organisms. There are numerous reports on the
potential use of biocontrol agents as replacements for
agrochemicals (Shimizu et al. 2000; Yang et al. 2007).
Actinomycetes are Gram-positive bacteria. They are the
most widely distributed group of microorganisms in nature.
They are also well known as saprophytic soil inhabitants
(Takisawa et al. 1993). Most actinomycetes in soil belong to
the genus Streptomyces (Goodfellow and Simpson 1987) and
75% of biologically active compounds are produced by this
genus. Actinomycetes occur in the plant rhizosphere soil and
produce active compounds (Suzuki et al. 2000). Attention
has been paid to the possibility that actinomycetes can pro-
tect roots by inhibiting the development of potential fungal
pathogens by producing enzymes which degrade the fungal
cell wall or producing antifungal compounds (Goodfellow
and Williams 1983). For example, Streptomyces sp. strain
5406 has been used in China to protect cotton crops against
soil-borne pathogens (Valois et al. 1996). Actinomycetes can
promote plant growth by producing promoters such as
indole-3-acetic acid (IAA) to help growth of roots or produce
siderophores to improve nutrient uptake (Merckx et al.
1987). However, the rate of discovery of new secondary
metabolites has been decreasing, so the discovery of acti-
nomycetes in several sources increases the chance for the
discovery of new secondary metabolites (Hayakawa et al.
2004). Active actinomycetes may be found in medicinal
plant root rhizosphere soils and may have the ability to
produce new inhibitory compounds.
S. Khamna � S. Lumyong (&)
Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand
e-mail: [email protected]; [email protected]
A. Yokota
Institute of Molecular and Cellular Biosciences,
The University of Tokyo, Tokyo, Japan
123
World J Microbiol Biotechnol (2009) 25:649–655
DOI 10.1007/s11274-008-9933-x
The present studies involved the isolation and identifi-
cation of actinomycetes from medicinal plant rhizosphere
soils. The isolates were characterized regarding their bio-
control activity and their in vitro production of active
compounds related to plant growth promotion.
Materials and methods
Sampling
Soil samples were collected from 16 medicinal plant rhi-
zospheres in Lumphun Province. The samples were air
dried at room temperature for 7 days. Soil pH was analyzed
according to the method of Suzuki et al. (2000).
Isolation of actinomycetes
One gram of each air-dried soil sample was treated in two
ways: pretreated with 6% yeast extract and 0.05% sodium
dodecylsulfate (SDS) (Hayakawa et al. 1988) or pretreated
with 1.5% phenol (Hayakawa et al. 2004). Humic acid
vitamin agar (HVA), oatmeal agar (OMA) and starch–casein
agar (SCA) pH 7.0 were used as selective media for isolation
of actinomycetes. All media were supplemented with 100 lg
nystatin/ml, 100 lg cycloheximide/ml and 50 lg nalidixic
acid/ml (Teachowisan et al. 2003). The plates were incu-
bated at 28�C for 4 weeks. Individual colonies were re-
grown at 28�C on ISP-2 agar for purification. The isolated
colonies were subcultured onto Hickey–Trener (HT) slants
and kept in 20% glycerol at -20�C as stock culture.
Characterization of actinomycete isolates
Morphological identification and chemotaxonomic
analyses
Purified isolates were identified to genus level according to
Bergey’s Manual of Determinative Bacteriology (Holt et al.
1994) after direct microscopic observation at (400 and
10009 magnification) of the vegetative and aerial myce-
lium developed as growth on cover slips buried in ISP-2
medium. Color of spore mass and diffusible pigment pro-
duction were visually estimated by using a color chart. Cell
wall diaminopimelic acid (A2 pm) and sugar isomer were
analyzed as described by Hasegawa et al. (1983).
DNA extraction, amplification and sequencing of the 16S
rDNA of Streptomyces sp.CMU-PA101, Streptomyces
CMU-SK126 and Streptomyces CMU-H009
Genomic DNA of Streptomyces CMU-PA101, Streptomy-
ces CMU-SK126 and Streptomyces CMU-H009, which
show high ability to inhibit five pathogenic fungi, produced
siderophores and IAA, were prepared according to a
modification of the CTAB method (Murray and Thompson
1980). PCR amplification of 16S rDNA was carried out
with a set of universal primers 27f and 1525r. The nucle-
otide sequences of the 16S rDNA obtained were subjected
to BLAST analysis with the NCBI database and submitted
to GenBank.
In vitro antagonistic bioassay
The actinomycete isolates were evaluated for their activity
towards five pathogenic fungi: Alternaria brassicicola (rose
apple anthracnose), Colletotrichum gloeosporioides (potato
dry rot), Fusarium oxysporum (Chinese cabbage leaf spot),
Penicillium digitatum (orange green mold) and Sclerotium
rolfsii (damping-off of balsam) by dual-culture in vitro
assay. These fungi were maintained on potato dextrose agar
(PDA) at room temperature and kept in a culture collection
at the Laboratory of Applied Microbiology, Department of
Biology, Faculty of Science, Chiang Mai University. Fun-
gal discs (8 mm diam.), 5 days old on potato dextrose agar
(PDA) at 28�C were placed at the center of PDA plates.
Two actinomycete discs (8 mm) 5 days old, grown on yeast
malt extract agar (YM) incubated at 28�C were placed on
opposite sides of the plates, 3 cm away from the fungal disc.
Plates without the actinomycete disc served as controls. All
plates were incubated at 28�C for 14 days and colony
growth inhibition (%) was calculated by using the formula:
C - T/C 9 100, where C is the colony growth of pathogen
in control, and T is the colony growth of pathogen in dual-
culture. All isolates were tested in triplicate.
Indole acetic acid (IAA) production
The production of IAA by 200 actinomycete isolates was
determined according to the method of Bano and Musarrat
(2003). The actinomycete discs (8 mm), grown on yeast malt
extract agar (YM) incubated at 28�C for 5 days, were inoc-
ulated into 5 ml YM broth containing 0.2% L-tryptophan and
incubated at 28�C with shaking at 125 rev/min for 7 days.
Cultures were centrifuged at 11,000 rev/min for 15 min. One
milliliter of the supernatant was mixed with 2 ml of Sal-
kowski reagent. Appearance of a pink color indicated IAA
production. Optical density (OD) was read at 530 nm using a
spectrophotometer. The level of IAA produced was esti-
mated by comparison with an IAA standard.
Screening for siderophore production
The actinomycete discs (8 mm), grown on YM agar incu-
bated at 28�C for 5 days were inoculated on CAS-substrates
with modified Gaus No.1 medium (MGs) (You et al. 2004)
650 World J Microbiol Biotechnol (2009) 25:649–655
123
and incubated at 28�C for 10 days. The colonies with
orange zones were considered as siderophore-producing
isolates. The functional groups of the siderophores were
determined. The active isolates (width of orange zone on
CAS plate[20 mm) were cultured on modified Gaus No.1
broth and incubated at 28�C with shaking at 125 rpm for
10 days. Catechol-type siderophores were estimated by
Arnow’s method (Arnow 1937) and hydroxamate sidero-
phores were estimated by the Csaky test (Csaky 1948).
Results and discussion
Actinomycete isolates from rhizosphere soils
From 16 medicinal plant rhizosphere soils, 445 isolates of
actinomycete were obtained (Table 1). About 89% of the
isolates were presumed to be in genus Streptomyces and
11% were identified to the genera Acitinomadura, Micro-
bispora, Micromonospora, Nocardia and Nonomurea.
Three isolates were unidentified. Streptomyces were pres-
ent in all rhizosphere soils, regardless of wild or
agricultural plant species, suggesting their wide distribu-
tion in association with plants in the natural environment.
The others actinomycetes were rare and could be isolated
from some rhizosphere soils. Streptomyces were the dom-
inant actinomycetes isolated from all 16 plant rhizosphere
soils, a result reported by others (Atalan et al. 2000;
Jayasinghe and Parkinson 2007; Pandey and Palni 2007;
Sembiring et al. 2000). The number and diversity of acti-
nomycetes isolated from Curcuma mangga rhizosphere
were higher than from other rhizosphere soils. Merckx
et al. (1987) indicated that the rhizosphere represents a
unique biological niche that supports abundant and diverse
saprophytic microorganisms because of a high input of
organic materials derived from the plant roots and root
exudates. Previous studies have shown that diversity of
actinomycetes in rhizosphere soils is positively correlated
to the level of organic matter and depended on the species
of plant (Germida et al. 1998; Hayakawa et al. 1988; Henis
1986). Tewtrakul and Subhadhirasakul (2007) found that
the roots of Curcuma mangga produced an antimicrobial
compound. It is possible that root exudates from this plant
might promote the growth of actinomycetes and antimi-
crobial compounds from the roots might decrease the
number of other soil bacteria and fungi so that the diversity
of actinomycetes from this soil is higher than other soils.
Effect of the pretreatment approach
The number of actinomycetes isolated from soils pretreated
with 6% yeast extract and 0.05% SDS was higher than
from those pretreated with 1.5% phenol. Pretreatment with
6% yeast extract and 0.05% SDS increased efficiency when
Table 1 Occurrence and distribution of actinomycetes from rhizosphere soils of medicinal plants
Plant rhizosphere soil pH No. of
StreptomycesNo. of rare actinomycete isolates
A B C D E F
Acanthus ebrateatus Vahl. (sea holly) 7.30 21
Achyranthes aspera L. (prickly chaff flower) 6.92 30 1 1
Amaranthus gracilis Desf. (spinach) 6.93 23 1
Bariena lunulina L. 6.98 17 2
Boesenbergia pandurata Schl. (fingerroot) 5.80 19 1
Curcuma mangga Val. and Zijp. 6.90 45 1 1 1 2 1 0
Cymbopogon citratus Stapf. (lemongrass) 7.00 29 2
Cymbopogon nardus Rendle.(citronellagrass)
7.00 13 3 1
Cyperus rotundus L. (cocograss) 6.38 10 1
Imperata cylindrical Beauv. (cogongrass) 6.92 30 1
Languas galanga L. (galangal) 6.89 22 2
Ocimum sanctum L. (holy basil) 7.01 32 1 4 1
Pandanus amaryllifolius Roxb.
(pandanus palm)
6.87 42 1 4
Rhinacanthus nasutus Kurz. 7.09 21 1 3
Stemona tuberosa Lour. (stemona) 6.93 18 1 1 4 1 2
Zingiber officinale Rose. (ginger) 6.63 24 1 2
Total 396 (89.0%) 4 (0.90%) 2 (0.50%) 6 (1.40%) 31 (7.0%) 3 (0.70%) 3 (0.70%)
A, Actinomadura; B, Microbispora; C, Micromonospora sp.; D, Nocardia sp.; E, Nonomurea sp.; F, unidentified
World J Microbiol Biotechnol (2009) 25:649–655 651
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isolating general actinomycetes (Hayakawa et al. 1988).
Phenol is a biocide and toxic to actinomycetes, so treat-
ment with 1.5% phenol reduced the number of
actinomycetes which were sensitive to this biocide
(Hayakawa et al. 2004). Humic acid vitamin agar was the
best medium for isolating actinomycetes from both pre-
treatments because this medium contained soil humic acid
as sole carbon and nitrogen sources which were suitable for
recovery of actinomycetes from soil samples (Fig. 1).
Antimicrobial activities
Twenty-three (5.2%) of actinomycete isolates were active
against at least one of the five pathogenic fungi. All the
active isolates were identified as Streptomyces sp.
(Table 2). Most of the active strains were isolated from
pandanus palm (Pandanus amaryllifolius) rhizosphere.
Lemanceau et al. (1995) and Wiehe et al. (1996) indicated
that differences in the quantitative and qualitative compo-
sition of root excretions provide different impact on the
rhizosphere microbiota and attract more or less bacterial
antagonists responsible for natural soil suppression. Plant
root exudates stimulate growth of rhizosphere actinomy-
cetes that are strongly antagonistic to fungal pathogens,
while the actinomycetes utilize root exudates for growth
and synthesis of antimicrobial substances (Crawford et al.
1993; Yuan and Crawford 1995). It is possible that
Fig. 1 Number of actinomycete isolates using two pretreatment
methods and three media
Table 2 Antifungal activities of Streptomyces isolates
Streptomycesisolates
% inhibitiona
Alternariabrassicicola
Colletotrichumgloeosporioides
Fusariumoxysporum
Penicilliumdigitatum
Sclerotiumrolfsii
CMU C14-12 0 0 55.4 ± 1.2 0 0
CMU Gin001 0 0 0 58.4 ± 0.8 0
CMU Gin003 26.5 ± 0.7 84.6 ± 0.6 68.7 ± 0.4 62.2 ± 0.5 0
CMU Gin005 0 0 0 62.3 ± 0.4 0
CMU G7-2 0 0 0 69.8 ± 0.2 0
CMU H001 0 0 0 58.7 ± 0.3 0
CMU PA001 0 0 25 ± 0.3 0 0
CMU PA101 97.5 ± 0.6 85.0 ± 0.4 74.2 ± 0.3 98.5 ± 0.8 77.5 ± 0.7
CMU PA510 0 0 25 ± 0.5 45.5 ± 0.8 0
CMU PA511 46.0 ± 0.4 42.9 ± 0.8 0 40 ± 0.9 0
CMU PA517 40.0 ± 0.6 57.1 ± 0.8 43.7 ± 0.9 0 0
CMU PA521 0 20.6 ± 0.6 0 63.9 ± 0.3 0
CMU PA528 0 0 0 42.5 ± 1.1 0
CMU PA531 0 0 0 44.0 ± 0.5 0
CMU PA529 0 28.6 ± 0.5 0 0 0
CMU PA537 49.0 ± 0.5 0 0 0 0
CMU PA533 0 42.9 ± 1.1 0 0 0
CMU PA539 0 42.9 ± 0.7 0 0 0
CMU SK126 69.9 ± 0.9 70.0 ± 0.5 77.5 ± 0.4 55.0 ± 0.2 68.8 ± 1.0
CMU SK132 0 0 0 39.9 ± 0.8 0
CMU UK102 0 0 25.0 ± 0.4 0 0
CMU W110 0 0 37.5 ± 0.3 0 0
CMU X209 0 0 25.0 ± 0.4 0 0
a Average ± standard error from triplicate samples
652 World J Microbiol Biotechnol (2009) 25:649–655
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excretions from the roots of pandanus palm might induce
actinomycetes that show anti-fungal activity. Two isolates,
Streptomyces CMU-PA101 (accession number FJ025786)
from P. amaryllifolius rhizosphere and Streptomyces
CMU-SK126 (accession number FJ217218) from
C. mangga rhizosphere, strongly inhibited all of the path-
ogenic fungi (Fig. 2). The 16SrRNA gene sequences of
Streptomyces CMU-PA101 and Streptomyces CMU-SK126
were similar to Streptomyces spectabilis (99% identity) and
Streptomyces cinnamoneus (99% identity). Similar studies
have been carried out by other workers. Ouhdouch and
Barakate (2001) found 10 isolates of actinomycetes from
medicinal plant rhizosphere soils, most of which were
Streptomyces spp. After testing for antifungal activity
against Candida albicans and C. tropicalis, they found that
all Streptomyces had antifungal activity. Thangapandian
et al. (2007) isolated Streptomyces from medicinal plant
rhizosphere soils and 8 isolates had antipathogenic activity.
Crawford et al. (1993) found that 12 actinomycete strains
isolated from Taraxicum officinale rhizosphere were active
against Pythium ultimum. Although 89.5% of the Strepto-
myces isolates in this study did not show any antifungal
activity towards the test organisms, they might produce
other useful compounds.
Fig. 2 Zones of growth inhibition caused by metabolites from
Streptomyces CMU PA101, grown on potato dextrose agar for
14 days, against a Fusarium oxysporum b Sclerotium sp. c Collet-otrichum gloeosporioides. Left, control; right, in the presence of
Streptomyces CMU PA101
Table 3 IAA production by actinomycete isolates after 7 days
incubation
Genus Isolates IAA production (lg/ml)a
Actinomadura CMU-AW310 17.44 ± 0.1
Actinomadura CMU-li5 5.47 ± 0.7
Actinomadura CMU-Li7 29.20 ± 0.4
Nocardia CMU-li6 44.73 ± 0.9
Nocardia CMU-O107 54.44 ± 0.2
Nonomurea CMU-AW311 31.71 ± 0.1
Streptomyces CMU-Aa104 57.46 ± 0.9
Streptomyces CMU-At204 29.22 ± 0.2
Streptomyces CMU-Aw312 30.66 ± 0.3
Streptomyces CMU-Bc014 16.93 ± 0.2
Streptomyces CMU-CL401 24.31 ± 0.3
Streptomyces CMU-Gin001 33.83 ± 06
Streptomyces CMU-Gin002 13.93 ± 0.4
Streptomyces CMU-Gin003 53.79 ± 0.5
Streptomyces CMU-Gin004 16.85 ± 0.9
Streptomyces CMU-Gin006 36.61 ± 0.2
Streptomyces CMUG-I 13.64 ± 0.5
Streptomyces CMU-H009 143.95 ± 0.2
Streptomyces CMU-H011 26.80 ± 1.8
Streptomyces CMU-K101 13.23 ± 0.2
Streptomyces CMU-K101 13.19 ± 0.3
Streptomyces CMU-K201 12.32 ± 1.8
Streptomyces CMU-K202 18.06 ± 0.3
Streptomyces CMU-K204 11.65 ± 0.7
Streptomyces CMU-L105 14.60 ± 0.5
Streptomyces CMU-PA101 28.86 ± 0.3
Streptomyces CMU-PA203 31.88 ± 0.2
Streptomyces CMU-PA301 19.09 ± 0.8
Streptomyces CMU-PA524 20.39 ± 0.7
Streptomyces CMU-PE401 28.52 ± 0.6
Streptomyces CMU-SK126 13.79 ± 0.3
Streptomyces CMU-T101 11.31 ± 1.6
Streptomyces CMU-T301 25.79 ± 0.3
Streptomyces CMU-VAN301 29.34 ± 0.4
Streptomyces CMU-VAN307 16.01 ± 0.3
Streptomyces CMU-X208 11.03 ± 0.2
a Average ± standard error from triplicate samples
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Indole acetic acid (IAA) and siderophore production
Thirty-six (8.1%) of actinomycete isolates produced IAA,
and 30 of these were Streptomyces sp. (Table 3). The
range of IAA production was 5.5–144 lg/ml. Streptomy-
ces CMU-H009 (accession number FJ185171) isolated
from lemongrass (Cymbopogon citrates) showed high
ability to produce IAA. The 16SrRNA gene sequence was
found to be 99% identical, to Streptomyces viridis. El-
Tarabilya and Sivasithamparamb (2006) and Tsavkelova
et al. (2006) found that Streptomyces from many crop
rhizosphere soils have the ability to produce IAA and
promoted plant growth. In the rhizosphere soils, root
exudates are the natural source of tryptophan for rhizo-
sphere micro-organisms, which may enhance auxin
biosynthesis in the rhizosphere. It is possible that high
tryptophan will be present in root exudates of lemongrass
and enhance IAA biosynthesis in Streptomyces CMU-
H009. Siderophore production were found in 45 (27.5%)
of all actinomycete isolates. The active isolates grew on
CAS agar and an orange halo formed around the colonies.
Most of them were Streptomyces (Table 4). Streptomyces
CMU-SK126 isolated from C. mangga rhizosphere soil
showed high ability to produce siderophores. This isolate
produced catechols 16.19 lg/ml and hydroxamate
54.9 lg/ml on modified Gaus No.1 broth (Table 5).
Usually, siderophores are produced by various soil
microbes to bind Fe3? from the environment, transport it
back to the microbial cell and make it available for
growth (Leong 1996; Neilands and Leong 1986). Micro-
bial siderophores may also be utilized by plants as an iron
source (Bar-Ness et al. 1991; Wang et al. 1993). Rhizo-
sphere soil actinomycetes have to compete with other
rhizosphere bacteria and fungi for iron supply and
therefore siderophore production may be very important
for their growth. Competition for iron is also a possible
mechanism to control the phytopathogens. Soil Strepto-
myces have been reported to produce hydroxamate-type
siderophores that could inhibit the growth of phytopath-
ogens by competition for iron in plant rhizosphere soils
(Muller et al. 1984; Muller and Raymond 1984; Tokala
et al. 2002).
From the present study, it could be demonstrated that
rhizosphere soil from Curcuma mangga provided a rich
source of diversity of actinomycetes. Streptomyces CMU-
PA101, Streptomyces CMU-SK126 and Streptomyces
CMU-H009 had the ability to produce high antifungal
compounds, siderophore and IAA. However, more detailed
investigation is required to demonstrate the potential of
these organisms for the biocontrol of pathogenic fungi and
in plant growth promotion which may be useful in phar-
macological and agricultural fields in the future.
Acknowledgments This work was supported by The Royal Golden
Jubilee Ph.D. Program (PHD/0153/2546). We are grateful to Dr. Eric
H. C McKenzie (Landcare Research, Private Bag 92170, Auckland,
New Zealand) for improving the English text.
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?, \10 mm; ??, 10–20 mm;
???, 21–30 mm; ????,
[30 mm
CAS-positive Genus
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