World J Microbiol Biotechnol (2009) 25:649655 DOI 10.

1007/s11274-008-9933-x

ORIGINAL PAPER

Actinomycetes isolated from medicinal plant rhizosphere soils: diversity and screening of antifungal compounds, indole-3-acetic acid and siderophore production
Sutthinan Khamna Akira Yokota Saisamorn 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. Morphological 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 unclassied. The highest number and diversity of actinomycetes were isolated from Curcuma mangga rhizosphere soil. Twenty-three Streptomyces isolates showed activity against at least one of the ve phytopathogenic fungi: Alternaria brassicicola, Collectotrichum 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
S. Khamna S. Lumyong (&) Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand e-mail: koymicro@yahoo.com; scboi009@chiangmai.ac.th A. Yokota Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

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 biological control agents have high potential to control plant pathogens and no effect on the environment or other nontarget 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 protect 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 actinomycetes 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.

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The present studies involved the isolation and identication of actinomycetes from medicinal plant rhizosphere soils. The isolates were characterized regarding their biocontrol 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 rhizospheres 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 starchcasein 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 incubated at 28C for 4 weeks. Individual colonies were regrown at 28C on ISP-2 agar for purication. The isolated colonies were subcultured onto HickeyTrener (HT) slants and kept in 20% glycerol at -20C as stock culture. Characterization of actinomycete isolates

show high ability to inhibit ve pathogenic fungi, produced siderophores and IAA, were prepared according to a modication of the CTAB method (Murray and Thompson 1980). PCR amplication of 16S rDNA was carried out with a set of universal primers 27f and 1525r. The nucleotide 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 ve 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. Fungal discs (8 mm diam.), 5 days old on potato dextrose agar (PDA) at 28C 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 28C 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 28C 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 dualculture. All isolates were tested in triplicate. Indole acetic acid (IAA) production

Morphological identication and chemotaxonomic analyses Puried isolates were identied to genus level according to Bergeys Manual of Determinative Bacteriology (Holt et al. 1994) after direct microscopic observation at (400 and 10009 magnication) of the vegetative and aerial mycelium developed as growth on cover slips buried in ISP-2 medium. Color of spore mass and diffusible pigment production 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, amplication and sequencing of the 16S rDNA of Streptomyces sp.CMU-PA101, Streptomyces CMU-SK126 and Streptomyces CMU-H009 Genomic DNA of Streptomyces CMU-PA101, Streptomyces CMU-SK126 and Streptomyces CMU-H009, which

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 28C for 5 days, were inoculated into 5 ml YM broth containing 0.2% L-tryptophan and incubated at 28C 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 Salkowski 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 estimated by comparison with an IAA standard. Screening for siderophore production The actinomycete discs (8 mm), grown on YM agar incubated at 28C for 5 days were inoculated on CAS-substrates with modied Gaus No.1 medium (MGs) (You et al. 2004)

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and incubated at 28C 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 modied Gaus No.1 broth and incubated at 28C with shaking at 125 rpm for 10 days. Catechol-type siderophores were estimated by Arnows method (Arnow 1937) and hydroxamate siderophores 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 identied to the genera Acitinomadura, Microbispora, Micromonospora, Nocardia and Nonomurea. Three isolates were unidentied. Streptomyces were present in all rhizosphere soils, regardless of wild or agricultural plant species, suggesting their wide distribution in association with plants in the natural environment. The others actinomycetes were rare and could be isolated from some rhizosphere soils. Streptomyces were the dominant 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 actinomycetes 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 antimicrobial 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 efciency when

Table 1 Occurrence and distribution of actinomycetes from rhizosphere soils of medicinal plants Plant rhizosphere soil pH No. of Streptomyces 21 30 23 17 19 45 29 13 10 30 22 32 42 21 18 24 396 (89.0%) 4 (0.90%) 2 (0.50%) 1 1 1 6 (1.40%) 1 1 1 1 2 4 4 3 4 2 31 (7.0%) 3 (0.70%) 3 (0.70%) 1 2 1 1 1 1 1 1 2 1 2 2 3 1 1 1 0 1 No. of rare actinomycete isolates A B C D E F

Acanthus ebrateatus Vahl. (sea holly) Achyranthes aspera L. (prickly chaff ower) Amaranthus gracilis Desf. (spinach) Bariena lunulina L. Boesenbergia pandurata Schl. (ngerroot) Curcuma mangga Val. and Zijp. Cymbopogon citratus Stapf. (lemongrass) Cymbopogon nardus Rendle. (citronellagrass) Cyperus rotundus L. (cocograss) Imperata cylindrical Beauv. (cogongrass) Languas galanga L. (galangal) Ocimum sanctum L. (holy basil) Pandanus amaryllifolius Roxb. (pandanus palm) Rhinacanthus nasutus Kurz. Stemona tuberosa Lour. (stemona) Zingiber ofcinale Rose. (ginger) Total

7.30 6.92 6.93 6.98 5.80 6.90 7.00 7.00 6.38 6.92 6.89 7.01 6.87 7.09 6.93 6.63

A, Actinomadura; B, Microbispora; C, Micromonospora sp.; D, Nocardia sp.; E, Nonomurea sp.; F, unidentied

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best medium for isolating actinomycetes from both pretreatments 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 ve pathogenic fungi. All the active isolates were identied 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 composition 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 actinomycetes 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

isolating general actinomycetes (Hayakawa et al. 1988). Phenol is a biocide and toxic to actinomycetes, so treatment 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
Table 2 Antifungal activities of Streptomyces isolates Streptomyces isolates % inhibitiona Alternaria brassicicola 0 0 26.5 0.7 0 0 0 0 97.5 0.6 0 46.0 0.4 40.0 0.6 0 0 0 0 49.0 0.5 0 0 69.9 0.9 0 0 0 0 Colletotrichum gloeosporioides 0 0 84.6 0.6 0 0 0 0 85.0 0.4 0 42.9 0.8 57.1 0.8 20.6 0.6 0 0 28.6 0.5 0 42.9 1.1 42.9 0.7 70.0 0.5 0 0 0 0

Fusarium oxysporum 55.4 1.2 0 68.7 0.4 0 0 0 25 0.3 74.2 0.3 25 0.5 0 43.7 0.9 0 0 0 0 0 0 0 77.5 0.4 0 25.0 0.4 37.5 0.3 25.0 0.4

Penicillium digitatum 0 58.4 0.8 62.2 0.5 62.3 0.4 69.8 0.2 58.7 0.3 0 98.5 0.8 45.5 0.8 40 0.9 0 63.9 0.3 42.5 1.1 44.0 0.5 0 0 0 0 55.0 0.2 39.9 0.8 0 0 0

Sclerotium rolfsii 0 0 0 0 0 0 0 77.5 0.7 0 0 0 0 0 0 0 0 0 0 68.8 1.0 0 0 0 0

CMU C14-12 CMU Gin001 CMU Gin003 CMU Gin005 CMU G7-2 CMU H001 CMU PA001 CMU PA101 CMU PA510 CMU PA511 CMU PA517 CMU PA521 CMU PA528 CMU PA531 CMU PA529 CMU PA537 CMU PA533 CMU PA539 CMU SK126 CMU SK132 CMU UK102 CMU W110 CMU X209
a

Average standard error from triplicate samples

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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 pathogenic 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 ofcinale rhizosphere were active against Pythium ultimum. Although 89.5% of the Streptomyces isolates in this study did not show any antifungal activity towards the test organisms, they might produce other useful compounds.

Table 3 IAA production by actinomycete isolates after 7 days incubation Genus Actinomadura Actinomadura Actinomadura Nocardia Nocardia Nonomurea Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces Isolates CMU-AW310 CMU-li5 CMU-Li7 CMU-li6 CMU-O107 CMU-AW311 CMU-Aa104 CMU-At204 CMU-Aw312 CMU-Bc014 CMU-CL401 CMU-Gin001 CMU-Gin002 CMU-Gin003 CMU-Gin004 CMU-Gin006 CMUG-I CMU-H009 CMU-H011 CMU-K101 CMU-K101 CMU-K201 CMU-K202 CMU-K204 CMU-L105 CMU-PA101 CMU-PA203 CMU-PA301 CMU-PA524 CMU-PE401 CMU-SK126 CMU-T101 CMU-T301 CMU-VAN301 CMU-VAN307 CMU-X208 IAA production (lg/ml)a 17.44 0.1 5.47 0.7 29.20 0.4 44.73 0.9 54.44 0.2 31.71 0.1 57.46 0.9 29.22 0.2 30.66 0.3 16.93 0.2 24.31 0.3 33.83 06 13.93 0.4 53.79 0.5 16.85 0.9 36.61 0.2 13.64 0.5 143.95 0.2 26.80 1.8 13.23 0.2 13.19 0.3 12.32 1.8 18.06 0.3 11.65 0.7 14.60 0.5 28.86 0.3 31.88 0.2 19.09 0.8 20.39 0.7 28.52 0.6 13.79 0.3 11.31 1.6 25.79 0.3 29.34 0.4 16.01 0.3 11.03 0.2

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 Colletotrichum gloeosporioides. Left, control; right, in the presence of Streptomyces CMU PA101

Streptomyces Streptomyces Streptomyces

a

Average standard error from triplicate samples

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654 Table 4 Siderophoreproducing actinimycete isolates

World J Microbiol Biotechnol (2009) 25:649655

CAS-positive Halo diameter ???? ??? ?? ?

Genus Streptomyces 6(3%) 4(2%) 8(4%) 20(10%) 136(68%) 174(87%) Actinomadura 0(0%) 1(0.5%) 0(0%) 2(1.0%) 1(0.5%) 4(2%) Microbispora 0(0%) 0(0%) 1(0.5%) 0(0%) 1(0.5%) 2(1%) Nocardia 0(0%) 0(0%) 3(1.5%) 1(0.5%) 16(8.0%) 20(10%)

?, \10 mm; ??, 1020 mm; ???, 2130 mm; ????, [30 mm

Not active Total

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 5.5144 lg/ml. Streptomyces 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. ElTarabilya 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 rhizosphere 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 CMUH009. 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 modied 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). Microbial siderophores may also be utilized by plants as an iron source (Bar-Ness et al. 1991; Wang et al. 1993). Rhizosphere 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 Streptomyces have been reported to produce hydroxamate-type siderophores that could inhibit the growth of phytopathogens by competition for iron in plant rhizosphere soils (Muller et al. 1984; Muller and Raymond 1984; Tokala et al. 2002).

Table 5 Siderophore production by active actinomycete isolates after 10 days Actinomycetes Actinomadura CMU-Y218 Streptomyces CMU-A104 Streptomycete CMU-AT204 Streptomyces CMU-GIN004 Streptomyces CMU-H009 Streptomyces CMU-K203 Streptomyces CMU-L206 Streptomyces CMU-PA101 Streptomyces CMU-SK107 Streptomyces CMU-SK126 Streptomyces CMU-VAN301
a

Catechols (lg/ml)a 3.94 0.9

Hydroxamate (lg/ml)a 20.0 0.2 52.42 0.3 6.97 0.6 10.00 0.5 32.73 0.9 12.42 0.7 26.97 1.0 21.82 0.4 25.76 0.3

7.84 0.1

16.19 0.5

54.85 1.2 14.85 0.7

Average standard error from triplicate samples

From the present study, it could be demonstrated that rhizosphere soil from Curcuma mangga provided a rich source of diversity of actinomycetes. Streptomyces CMUPA101, 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 pharmacological and agricultural elds 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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