Isolation of endophytic actinomycetes from selected plants and their antifungal

activity

Thongchai Taechowisan1, John F. Peberdy2 and Saisamorn Lumyong1,*1Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand2School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK*Author for correspondence: Tel.: +66-53-943346 Ext: 1503, Fax: +66-53-892259,E-mail: scboi009@chiangmai.ac.th

Received 21 August 2002; accepted 20 December 2002

Keywords: Actinomycetes, antifungal activity, endophytes, Micromonospora, Nocardia, Streptomyces

Summary

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humicacid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leavesand 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophyticactinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Mostisolates were classified as Streptomyces sp. (n ¼ 277); with the remainder belonging to Microbispora sp. (n ¼ 14),Nocardia sp. (n ¼ 8) and Micromonospora sp. (n ¼ 4). Four isolates were unclassified and 23 were lost duringsubculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samplesof Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibitedColletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of bothtest fungi.

Introduction

Actinomycetes are prokaryotes which have a hyphal(hence fungal-like) morphology. Most of the actinomy-cetes described are soil microorganisms and are active inthe decomposition of plant tissues, and thereby in therecycling of carbon and nitrogen. Members of the genusFrankia are an exception, as these bacteria exist both asfree-living forms and as endophytes forming nodules onhost plants (Baker et al. 1980; Knowlton et al. 1980).However, these reports give no indication of prevalenceof different species growing as endophytes in thedifferent tissues of their host plants. Several reportsrefer to actinomycete activity in the protection of theplant host against pathogens and the influence of theirmetabolic products on plant growth and physiology(Katznelson & Cole 1965; Tahvonen 1982; Williamset al. 1984; Drautz & Zahner 1986; Schippers et al.1987). Other reports refer to pathological interactions ofendophytic actinomycetes with plants (Alwadi & Baka2000).We are interested in the antifungal activity of endo-

phytic actinomycetes which has been a focus in theexploitation of these organisms as excellent biocontrolagents against phytopathogenic fungi. To date, how-ever, much less is known about the antifungal antibiot-

ics produced by endophytic actinomycetes (Sardi et al.1992). The present study involved the isolation ofactinomycetes from the tissues of healthy plants, aninitial identification of them and an evaluation of theantifungal activity of their secondary metabolites.

Materials and methods

Sample collection

Leaf, stem and root tissues were recovered from healthyrepresentatives of herbaceous and woody plants fromthe environs of Chiang Mai, Thailand during the periodSeptember, 2001–February, 2002.

Isolation of actinomycetes

The samples were dissected into leaves, stems and roots,washed in running tap water and cut into small piecesof ca. 4� 4 mm2. Tissue pieces were rinsed in 0.1%Tween20 for 30 s, then in 1% sodium hypochlorite for5 min, and then washed in sterile distilled water for5 min. Next the tissue pieces were surface sterilized in70% ethanol for 5 min and air-dried in a laminar flowchamber. Finally the pieces were transferred to dishes of

World Journal of Microbiology & Biotechnology 19: 381–385, 2003. 381� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

humic acid–vitamin (HV) agar (Otoguro et al. 2001)containing 100 lg nystatin and cycloheximide/ml, andincubation at 30 �C was continued for about 1 month.The colonies were inoculated onto to ISP-2 medium(Shirling & Gottlieb 1966) for purification. The isolatedcolonies were subcultured onto a Hickey–Tresner (HT)medium (Redshow et al. 1976) slants to establish stockcultures. Isolate prevalence was calculated as follows(Bussaban et al. 2001)

Isolate prevalence

¼ Number of samples yielding one isolate� 100

Number of samples in that trial

Morphological observations

Leaf, stem and root materials from Zingiber officinale L.(Zingiberaceae) and Alpinia galanga L. (Zingiberaceae)were selected for microscopic observation by scanningelectron microscopy (SEM) (JEOL-JSM840A SEM,Tokyo, Japan). Specimens were washed several timesusing distilled water and fixed overnight in 2.5%glutaraldehyde at 4 �C. They were then dehydrated ina graded alcohol series (30–95%) followed by treatmentin acetone and critical-point drying (Petrolini et al.1986). The specimens from each process were mountedon stubs, splutter-coated with gold, and viewed on theSEM at an accelerating voltage of 20 kV. Photomicro-graphs were recorded on Kodak VP200 film (New York,USA).

Taxonomic properties

Methods and media described by the InternationalStreptomyces Project (Shirling & Gottlieb 1966) wereused to determine most of the cultural and physiologicalcharacteristics. For morphological characteristics, thepresence of aerial mycelium, spore mass colour, distinc-tive reverse colony colour, diffusible pigment, andsporophore and spore chain morphology were recordedafter 10 days incubation on ISP-2 medium. Diamino-pimelic acid isomers and sugars from whole-cell extractwere analysed for chemotaxonomic studies (Beckeret al. 1964; Boone & Pine 1968).

Antifungal activity of actinomycetes isolates againstphytopathogenic fungi

The endophytic isolates were cultured on plates on ISP-2. Two fungal pathogens Colletotrichum musae andFusarium oxysporum, the causative agents of anthrac-nose of banana and wilt of wheat, respectively, wereused for screening antifungal activity. They were grownon potato dextrose agar (PDA). Mycelial disks of 6 mmdiameter were cut from the plates with the two patho-gens and transferred to the ISP-2 plates and positioned6 cm away from each pre-grown actinomycete colony.

The plates were incubated at 30 �C for 5–7 days. Thewidth of inhibition zones between the pathogen and theactinomycete isolates was measured and evaluated asfollows: þþþ, 20 mm<; þþ, 11–19 mm; +, 2–10 mm; ±, £1 mm; ), 0 mm.

Results and discussion

After 3–4 weeks incubation, the surface of some tissuesamples showed hyphal growth which subsequentlygrew out onto the surface of the HV agar (Figure 1a).This process of growth of the actinomycetes through thesurfaces of the tissues was observed by SEM (Fig-ure 1b). Growth of bacteria and fungi from the tissueswas almost completely inhibited by the antibioticsincluded in HV agar leaving the actinomycetes clearlyvisible. The low level of bacterial contamination ob-

Figure 1. Growth of actinomycete colonies from sterilized blocks of

plant tissue on HV agar. (a) This plate was photographed after 3 weeks

of incubation. (b) Scanning electron micrograph of aerial hyphae of

actinomycetes which have grown through the epidermis of a leaf of A.

galanga. Magnification: 6500·.

382 T. Taechowisan et al.

served was due to Bacillus spp. This contamination mayhave arisen from spores on the surface of these tissuesthat were resistant to chemical surface sterilization ormay be due to an endophytic Bacillus sp. (Garbeva et al.2001; Bai et al. 2002). Incubation of surface-sterilizedplant parts in a moist chamber and plating of planttissues on agar media are techniques usually employedin plant pathology, and not often used in microbialecology. However, they may be extremely useful in theisolation of microorganisms from unusual habitats.Using these techniques, we were able to confirm thepresence of endophytic actinomycetes in plant tissues,especially roots, where a large number of these organ-isms are most probably found. The actinomycete iso-lates took at least 3 weeks to grow out from the tissues.If the tissue sterilization procedure used in this studywas not sufficient to kill surface microbes, they would beexpected to grow from specimens within a few days.Some 36 plant species from the families Acanthaceae,

Amaranthaceae, Cruciferae, Cyperaceae, Gramineae,Iridaceae, Labiatiae, Rubiaceae, Rutaceae, Taccaceae,Umbelliferae and Zingiberaceae (Table 1), were exam-

ined using a total of 5400 each of root, stem and leaftissues. Streptomycetes were the most common isolatesrecovered, being most prevalent from roots (3.9%),leaves (1.8%) and less from stems (0.3%). The myceliaof the actinomycete isolates grew out of the tissue blocksonto the surfaces. Thus, these isolates are consideredendophytic rather than ectophytic microbes, as dis-cussed by Okazaki et al. (1995). With SEM, hyphae ofthese organisms could be recognized in the leaf and roottissues of both Zingiber officinale and Ailpinia galanga.Observations on 40 samples of each tissue type con-firmed the high incidence in roots (7.5%) and leaves(5%), but hyphae were not seen in stems. It is cleartherefore that roots present a good habitat for theseendophytic actinomycetes. However, the frequency re-ports from the SEM observations were higher suggest-ing that the visual observation revealed non-viablehyphae or organisms that could not grow on HV agar,which like all media will be selective according to itsnutrient availability. Future studies should thereforeinvolve the use of several media for isolation. Theisolates were obtained most frequently from roots and

Table 1. Numbers of isolates of endophytic actinomycete per tissue block from leaves, stems and roots or a range of herbaceous and woody

plants.

Family Host plant Leaves (%) Stems (%) Roots (%) Total (%)

Acanthaceae Rhinacanthus communisa 1 (0.6) 0 (0.0) 0 (0.0) 1 (0.2)

Amaranthaceae Amaranthus gracilisa 0 (0.0) 0 (0.0) 4 (2.6) 4 (0.8)

Cruciferae Brassica junceaa 0 (0.0) 0 (0.0) 1 (0.6) 1 (0.2)

Cruciferae Brassica oleraceaa 0 (0.0) 0 (0.0) 20 (13.3) 20 (4.4)

Cyperaceae Cyperus difformisa 6 (4.0) 3 (2.0) 4 (2.6) 13 (2.8)

Cyperaceae Cyperus iriaa 3 (2.0) 1 (0.6) 5 (3.3) 9 (2.0)

Cyperaceae Cyperus kyllingiaa 5 (3.3) 3 (2.0) 5 (3.3) 13 (2.8)

Cyperaceae Cyperus malaccensisa 3 (2.0) 5 (3.3) 5 (3.3) 13 (2.8)

Cyperaceae Cyperus rotundusa 5 (3.3) 1 (0.6) 4 (2.6) 10 (2.2)

Gramineae Chloris barbataa 0 (0.0) 2 (1.3) 0 (0.0) 2 (0.4)

Gramineae Cymbopogon citratusa 3 (2.0) 0 (0.0) 3 (2.0) 6 (1.3)

Gramineae Cymbopogon nardusa 3 (2.0) 0 (0.0) 9 (6.0) 12 (2.6)

Gramineae Echinochloa colonaa 3 (2.0) 0 (0.0) 1 (0.6) 4 (0.8)

Gramineae Echinochloa crusgallia 1 (0.6) 1 (0.6) 3 (2.0) 5 (1.1)

Gramineae Imperata cylindricaa 0 (0.0) 0 (0.0) 8 (5.3) 8 (1.7)

Iridaceae Eleutherine palmifoliaa 0 (0.0) 0 (0.0) 6 (4.0) 6 (1.3)

Labiatae Ocimum tenuifloruma 5 (3.3) 4 (2.6) 0 (0.0) 9 (2.0)

Rubiaceae Coffea arabicab 2 (1.3) 0 (0.0) 2 (1.3) 4 (0.8)

Rutaceae Citrus hystrixb 1 (0.6) 0 (0.0) 0 (0.0) 1 (0.2)

Taccaceae Tacca chantrieria 0 (0.0) 0 (0.0) 2 (1.3) 2 (0.4)

Umbelliferae Apium graveolensa 9 (6.0) 0 (0.0) 0 (0.0) 9 (2.0)

Umbelliferae Coriandrum sativuma 0 (0.0) 0 (0.0) 2 (1.3) 2 (0.4)

Zingiberaceae Alpinia blepharocalyxa 2 (1.3) 1 (0.6) 3 (2.0) 6 (1.3)

Zingiberaceae Alpinia galangaa 9 (6.0) 0 (0.0) 32 (21.3) 41 (9.1)

Zingiberaceae Amomum siamensea 10 (6.6) 0 (0.0) 28 (18.6) 38 (8.4)

Zingiberaceae Boesenbergia pandurataa 4 (2.6) 0 (0.0) 19 (12.6) 23 (5.1)

Zingiberaceae Curcuma domesticaa 3 (2.0) 0 (0.0) 10 (6.6) 13 (2.8)

Zingiberaceae Curcuma longaa 8 (5.3) 0 (0.0) 0 (0.0) 8 (1.7)

Zingiberaceae Etlingera elatiora 0 (0.0) 0 (0.0) 2 (1.3) 2 (0.4)

Zingiberaceae Zingiber cassumunara 3 (2.0) 0 (0.0) 2 (1.3) 5 (1.1)

Zingiberaceae Zingiber officinalea 8 (5.3) 0 (0.0) 33 (22.0) 40 (8.8)

Totals 97 (1.7) 21 (0.3) 212 (3.9) 330 (2.0)

Sterilized tissue blocks were placed on HV agar and incubated for up to a month at 30 �C.No isolates were recovered from the tree species, Citrus aurantifoliab (Rutaceae), Streblus asperb (Moraceae), Tamarindus indicab

(Leguminosae), Mangifera indicab (Anacardiaceae) and Dimocarpus longanb (Sapindaceae).a herbaceous plants; b woody plants.

Isolation of endophytic actinomycetes 383

less so from other parts. This may relate to the presenceof actinomycetes as a large part of the rhizospheremicrobial flora (Sardi et al. 1992) thus enabling easierinfection of a host. However, the presence of endophyticactinomycetes in leaves and stems support previousreports (Okazaki et al. 1995; Shimizu et al. 2000).The presence of endophytic actinomycetes, as shown

by their isolation from healthy plants, and the SEMinvestigations on internal tissues, leads to the conclusionthat there is a close relationship between these micro-organisms and plant tissues, in which growth of theformer could have a favourable effect on plant growthand development. Their biological activities can affectplant growth either through affecting the nutrient supply(Katznelson & Cole 1965; Tahvonen 1982; Williamset al. 1984; Drautz & Zahner 1986; Schippers et al.1987) or the in situ production of secondary metaboliteswhich stimulate or depress vegetative development(Mishra et al. 1987) and may also protect againstphytopathogenic microorganisms (Abd-Allah 2001;Getha & Vikineswary 2002).In this study most of the actinomycetes were obtained

from herbaceous plants and very few from woodyplants. Similar observations can be drawn from otherworkers. Okazaki et al. (1995) obtained 246 isolatesfrom 172 samples of healthy leaves of monocotyledonssuch as Cyperus sp. and Carex sp. and in comparisonShimizu et al. (2000) obtained 10 isolates from Rhodo-dendron sp. In our experiment the use of only onemedium for isolation of actinomycetes may be a factor,however, there are also intrinsic differences betweenwoody and herbaceous species and it maybe to thesethat we have to look for an explanation. Many treespecies have mycorrhizal fungi associated with theirroots which may form a barrier to infection of thesetissue by other endophytic species.In total 330 isolates were recovered, the majority of

which were Streptomyces spp., with the remainderidentified as Microbispora sp., Nocardia sp. and Micro-monospora sp. (Figure 2). Four isolates did not developsporing structures, although meso-diaminopimelic acidwas detected in whole cell extracts, confirming anactinomycete status. Correspondingly the prevalence ofStreptomyces sp. was the highest, ranging from 6.4% for

Z. officinale to 0.2% for Brassica juncea (Cruciferae) andfor Citrus hystrix (Rutaceae). Values of Microbisporasp., Nocardia sp. and Micromonospora sp. were muchlower (Table 2). These results indicate that herbaceousplants are the major host for endophytic actinomycetes,with Streptomyces spp. being dominant. In contrast, thisstudy has shown that actinomycetes are found onlyrarely in tree species such as Citrus aurantifolia (Ruta-ceae), Dimocarpus longana (Sapindaceae), Streblus asper(Moraceae), Tamarindus indica (Leguminosae) andMangifera indica (Anacardiaceae).The antifungal activity of endophytic actinomycete

isolates is shown in Table 3. The majority of the isolates(>200) appeared not to produce secondary metaboliteswhich displayed antifungal activity against the two testfungi. The remaining isolates could be divided into fivecategories according to the size of the growth-inhibitionzones produced. This survey revealed that only a smallnumber were strongly inhibitory to C. musae and F.oxysporum (Figure 3). In a similar study, Sardi et al.(1992) obtained ca. 500 isolates from the roots of 13plant species and most of these were Streptomyces sp.They classified these isolates into 72 groups based ontheir characteristics. After testing antimicrobial activity

Figure 2. The frequency of different actinomycete types isolated from

all the plant types investigated.

Table 2. The highest prevalence of actinomycete isolates and the

specific plants, based on isolations made on HV agar.

Isolatesa Host plant Highest prevalence

Streptomyces sp. Zingiber officinale 6.44%

Microbispora sp. Alpinia galanga 0.66%

Nocardia sp. Cyperus malaccensis 0.44%

Micromonospora sp. Alpinia galanga,

Boesenbergia pandurata,

Curcuma domestica and

Echinochloa colona

0.22%

a The most frequently isolated actinomycetes from the specific

plants.

Table 3. Antifungal activity of endophytic actinomycetes isolates

against C. musae and F. oxysporum.

Potential

antifungal

activity

Number of endophytic actinomycetes isolates (%)

against tested fungi

Colletotrichum

musae

Fusarium

oxysporum

Colletotrichum

musae and Fusarium

oxysporum

þþþþa 3 (0.9%)b 5 (1.5%)b 2 (0.6%)b

þþþ 10 (3.0%) 18 (5.4%) 8 (2.4%)

þþ 44 (13.3%) 53 (16.0%) 36 (10.9%)

þ 10 (3.0%) 16 (4.8%) 8 (2.4%)

Not active 240 (72.7%) 215 (65.1%) 253 (76.6%)

The potential of antifungal activity was evaluated by the zone of

fungal growth inhibition on ISP-2 medium after incubation at 30 �Cfor 7 days.a þþþþ: Width of growth inhibition zone >20 mm.

þþþ: 10–20 mm.

þþ: 1–10 mm.

+: <1 mm.b These isolates were identified to be Streptomyces sp.

384 T. Taechowisan et al.

of 10 groups against Micrococcus luteus and F. oxyspo-rum, then found that all groups had antimicrobialactivity against one or the other organisms, but not toboth. Thus most of their isolates had a narrow anti-microbial spectrum. From the present study results of invitro antifungal activity (Table 3), only two endophyticactinomycetes isolates had a strong potential of anti-fungal activity to Colletotrichum musae and Fusariumoxysporum. These results demonstrated that some ofendophytic actinomycetes have the potential for inhi-biting the growth of tested phytopathogenic fungi.However, more detailed investigation is required todemonstrate the potential of these organisms in thebiocontrol of plant diseases.

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Isolation of endophytic actinomycetes 385