Antonie van Leeuwenhoek 78: 353366, 2000. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

353

Selective isolation and characterisation of members of the Streptomyces violaceusniger clade associated with the roots of Paraserianthes falcataria
Langkah Sembiring, Alan C. Ward & Michael Goodfellow
Department of Agricultural and Environmental Science, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK ( Author for correspondence; E-mail: m.goodfellow@ncl.ac.uk) Key words: polyphasic taxonomy, rugose-ornamented spores, streptomycetes, Streptomyces violaceusniger clade

Abstract Large numbers of putatively novel streptomycetes were isolated from environmental samples collected from in and around the root system of the tropical angiosperm, Paraserianthes falcataria. Representative isolates were assigned to 37 multi-membered and 107 single membered colour groups based on their ability to form pigments on oatmeal and peptone yeast extract iron agars. The largest taxon, colour group 3, encompassed 94 isolates which had morphological properties typical of members of the Streptomyces violaceusniger clade. Twelve representatives of this taxon chosen on the basis of Curie-point pyrolysis mass spectrometric data were compared with representatives of the validly described species which constitute the Streptomyces violaceusniger clade. Six out of the twelve representative strains were readily distinguished from one another and from the marker strains using a combination of genotypic and phenotypic properties. These organisms were consequently considered to merit species status as Streptomyces asiaticus sp. nov., Streptomyces cangkringensis sp. nov., Streptomyces indonesiensis sp. nov., Streptomyces javensis sp. nov., Streptomyces rhizosphaerius sp. nov. and Streptomyces yogyakartensis sp. nov.

Introduction

Actinomycetes are an integral part of the indigenous soil microora involved in the turnover of recalcitrant plant organic matter but are generally seen to have a lesser role in the nutrient rich rhizosphere (Williams et al. 1984; Marilley & Aragno 1999). Nevertheless, there is evidence that actinomycetes, notably streptomycetes, are widely distributed in the root environment (Miller et al. 1990; Sardi et al. 1992; Crawford et al. 1993; Atalan et al. 2000) though little is known about the extent of their taxonomic diversity, ecosystem functions or interactions with other organisms therein. Some streptomycetes, notably members of the Streptomyces violaceusniger clade (Sembiring et al. 2000), are known to be antagonistic to several classes of plant pathogenic fungi (Trejo-Estrada et al. 1998a, b; Al-Tai et al. 1999). It has also been shown that streptomycetes isolated from rhizosphere soil represent novel species (Upton 1994; Atalan et al. 2000), members of which inhibit fungal pathogens in vitro and in vivo (Upton 1994).

Biosystematic studies on the actinomycete community in soil have been hampered by problems associated with representative sampling and poor systematics. However, the dispersion and differential centrifugation (DDC) technique, a multistage extraction procedure introduced by Hopkins et al. (1991a), has been shown to be effective for representative sampling of bacteria, including actinomycetes, from a range of soils (Hopkins et al. 1991b; MacNaughton & ODonnell 1994). Atalan et al. (2000) found that representative sampling of cultivable streptomycetes from rhizosphere soil can be achieved using the DDC technique coupled with the use of selective isolation procedures. They also showed that representatives of different Streptomyces species can be isolated at successive stages of the DDC procedure. It is still difcult to classify streptomycetes below the genus level due to the complexity of the taxon. However, Atalan et al. (2000) developed a workable strategy for determining the species richness of cultivable streptomycetes isolated from natural habitats; this involves the assignment of isolates to groups based on traditional pigmentation properties, and the evaluation

354 of the taxonomic integrity of these groups by Curie point pyrolysis mass spectrometry followed by more exacting molecular systematic studies on representatives of the pyrogroups. A similar strategy was used to detect putatively novel rhodococci that were selectively isolated from deep sea sediments in the N.W. Pacic Ocean (Colquhoun et al. 1998a, b, 2000). The present study was designed to determine the composition of cultivable members of the streptomycete community associated with the root system of the tropical angiosperm, Paraserianthes falcataria prior to conducting a polyphasic taxonomic study on isolates found to produce spiral chains of rugose ornamented spores. In addition, the effectiveness of a modied DDC and a tumble shaking procedure in extracting streptomycete propagules from composite environmental samples were compared. sions were added to 9 ml of 1/4 strength Ringers solution and the preparations shaken on a tumble shaker (model TM7-tumbler, Luckham Ltd., Burgess Hill, England, UK), at speed setting 4, at room temperature. The three 101 dilutions were then treated in the same way as the corresponding initial fractions prepared in the DDC procedure. All of the isolation plates were dried for 15 min prior to inoculation, as recommended by Vickers & Williams (1987). The inoculated plates, 3 per dilution, were incubated at 25 C for 14 days. Counts of presumptive streptomycetes growing on the starch casein agar plates were expressed as the mean number of colony forming units (cfu) per gram dry weight soil; dry weights were obtained after overnight treatment of the environmental samples at 105 C. Five hundred and fty six representatives of isolates putatively assigned to the genus Streptomyces on the basis of colony morphology, notably aerial spore mass colour, substrate mycelial pigmentation and the colour of any diffusible pigments, were subcultured onto modied Bennetts agar (Jones 1949), incubated at 25 C for 14 days, and checked for purity by microscopic examination of Gram-stained smears. The isolates were maintained as suspensions of spores and mycelial fragments in glycerol (20%, v/v) at 20 C and on modied Bennetts agar slopes (Jones 1949) at room temperature. Colour grouping and maintenance of strains. The representative isolates were inoculated onto oatmeal (ISP3; Kster 1959) and peptone-yeast extract-iron agar plates (ISP6; Shirling & Gottlieb 1966) which were incubated at 25 C for 14 and 4 days, respectively. The oatmeal agar plates were examined by eye and aerial spore mass colour, substrate mycelium pigmentation and colour of any diffusible pigments recorded using the National Bureau of Standards (NBS) Colour Name Charts (Kelly 1958; NBS 1964). The peptone-yeast extract-iron agar plates were used to determine whether the isolates produced melanin pigments. The isolates were assigned to 37 multimembered and 105 single membered groups, the largest of which contained 94 isolates (Table 1). The latter were considered to have colonial properties consistent with their assignment to the S. violaceusniger clade (Sembiring et al. 2000). Pyrolysis mass spectrometry. The 94 isolates presumptively assigned to the S. violaceusniger clade were grown, from glycerol stocks, for 7 days at 25 C on a non-sporulating agar (NSA) medium designed to

Materials and methods Sampling site and preparation of samples. Rhizosphere and non-rhizosphere samples were collected from, and around, the root system of the tropical angiosperm Paraserianthes falcataria in a reforested area at Cangkringan, near Yogyakarta, Java, Indonesia. Soil was shaken from the root material to give the rhizoplane sample. Selective isolation methods. Actinomycetes were isolated from composite non-rhizosphere, rhizosphere and rhizoplane samples using a modication of the DDC method (Hopkins et al. 1991b), and a tumble shaking technique. Mechanical blending of soil suspensions prepared from 5 grams of the soil, the initial step in the DDC procedure, was performed with an Ultra-Turax T25 homogeniser (Janke & Kunkel, Germany) using an 8 mm diameter probe (S25-86; Ultra-Turax). The four supernatant fractions derived from the DDC procedure were centrifuged separately at 12,000 g for 20 minutes at 4 C, suspended in 9 ml of 1/4 strength Ringers solution and the resultant 101 dilutions heated at 55 C for 20 min. The heat treated supernatant fractions were serially diluted in 1/4 strength Ringers solution down to 106 and aliquots (0.1 ml) of the resultant preparations used to inoculate plates of rafnose-histidine (pH 7.07.2; Vickers et al. 1984) and starch casein agars (Kster & Williams 1964) supplemented with cycloheximide and nystatin, each at 50 g ml1 . For the tumble shaking experiments, one gram of each of the three suspen-

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Table 1. Source of isolates putatively identied as members of the Streptomyces violaceusniger clade

356 inhibit sporulation of streptomycetes (Sanglier et al. 1992). In addition, 10 duplicated cultures were prepared. Growth from the plates was spread over sterile polycarbonate lters (0.45 m, 47 mm. diam., Millipore) placed on NSA plates which were incubated as before. Curie point PyMS analyses were made in triplicate on whole streptomycete samples (Ferguson et al. 1997) under vacuum at 530 C for 2.4 seconds (temperature rise time 0.6 s), using an Horizon RAPyD 400 spectrometer (Horizon Instruments Ltd., Heatheld, UK). The resultant data were analysed using established procedures (Kay et al. 1994; Trujillo & Goodfellow 1997). Phenotypic characterisation. Twelve isolates taken to represent the organisms presumptively assigned to the S. violaceusniger clade were compared with representatives of this taxon (Figure 3) for a range of phenotypic tests (Table 4) that have been described previously (Sembiring 2000; Sembiring et al. 2000). The phenotypic data were in one of two mutually exclusive states that were scored plus (1) or minus (0). The resultant dataset was analysed using the MultiVariate Statistical Package Plus-Version 2 (Kovach 1988) using the simple matching coefcient (SSM ; Sokal & Michener 1958). Clustering was achieved using the unweighted pair group method with arithmetic averages algorithm (UPGMA; Sneath & Sokal 1973) and the results presented as a dendrogram. The nal data matrix contained differential information on 21 strains and 32 unit characters. Detection of chemical and morphological markers. Biomass of the 12 representative isolates (Table 1) was prepared from modied Bennetts agar plates (Jones 1949) incubated at 25 C for 14 days. The isomeric forms of diaminopimelic acid (A2pm ) were determined by thin-layer chromatography of wholeorganism hydrolysates on cellulose acetate sheets after Staneck & Roberts (1974). Spore chain morphology and spore surface ornamentation were recorded using the procedures described by Sembiring et al. (2000). DNA preparation, amplication and determination of 16S rDNA sequences. DNA was extracted from the representative strains and amplied following the procedure used by Sembiring et al. (2000). The amplied samples were separated by gel electrophoresis, puried using a Nucleospin Extraction Kit (Biogen Ltd.) and sequenced directly using an ABI PRISMR Big DyeT M Terminator Cycle Sequencing Kit (Applied Biosystems) and previously described oligonucleotide primers (Chun & Goodfellow 1995). Sequencing gel electrophoresis was carried out and the nucleotide sequences automatically obtained by using an Applied Biosystems DNA sequencer (model 377) and software provided by the manufacturer. Phylogenetic analysis. The 16S rDNA sequences were aligned manually with available streptomycete nucleotide sequences retrieved from the Ribosomal Database Project (Maidak et al. 1997) and EMBL/GenBank databases by using the AL 16S (Chun 1995) and CLUSTAL X (Thompson et al. 1997) programs. This data set was examined using three treeing methods, namely the least-squares (Fitch & Margoliash 1967), maximum-likelihood (Felsenstein 1981) and neighbour-joining (Saitou & Nei 1987) algorithms, and the resultant unrooted tree topologies evaluated as described by Sembiring et al. (2000). The root position on the neighbour-joining tree was estimated using Nocardiopsis alborubidus DSM 40467T (accession number X97882) as the outgroup. Results Selective isolation, enumeration and colour grouping Isolates presumptively assigned to the genus Streptomyces were distinguished from other bacterial colonies growing on the rafnose-histidine and starchcasein isolation plates by their characteristic colonial and pigmentation properties. Large numbers of the target organisms were isolated from the heat treated suspensions of rhizosphere, rhizoplane and non-rhizosphere soils following incubation on the two selective isolation media. Higher presumptive streptomycete counts were consistently found on the starchcasein isolation plates seeded with fractions obtained using the DDC as opposed to the tumble shaking procedure (Table 2). The highest presumptive streptomycete counts were derived from the supernatant 1 fractions and the lowest from the supernatant 4 suspensions. The rhizosphere effect was more pronounced on the starch-casein plates seeded with fractions derived from the various stages of the DDC procedure. The representatives of the different kinds of presumptive streptomycetes growing on the rafnosehistidine and starch-casein isolation plates seeded with fractions obtained from the DDC procedure were assigned to 37 multi-membered (451 isolates) and 105

357
Table 2. Numbers of presumptive Streptomyces strains ( 104 cfu g1 weight soil) growing on starch casein isolation plates seeded with supernatant and residue fractions derived from the application of the dispersion and differential centrifugation technique, and suspensions obtained from tumble shaking, arising from composite soil samples associated with the root system of Paraserianthes falcataria Extraction technique Dispersion and differential tumple shaking centrifugation (DDC) (TS) 51.0 30 20.0 2.2 17.8 0.9 4.8 4.0 93.6 37.1 Numbers derived from DDC technique/numbers from TS

Fraction

Rhizosphere sample Supernatant 1 2 3 Residue Total Rhizoplane sample Supernatant 1 2 3 Residue

32.3 10.5

2.9

18.1 1.3 14.7 0.5 11.1 1.8 1.3 0.1 45.2 3.7 12.8 1.6 9.4 1.0 10.5 1.7 0.7 0.01 33.4 4.31

17.7 5.0

2.5

Non-rhizosphere sample Supernatant 1 2 3 Residue

24.5 15.6

1.36

The starch casein plates were supplemented with antifungal antibiotics and incubated at 25 C for The four fractions were obtained following treatment of the composite soil samples using a

14 days.

procedure slightly modied from the original dispersion and differential centrifugation procedure described by Hopkins et al. (1991b).

single membered colour groups. In the corresponding experiment involving propagules obtained from the tumble shaking fractions 51 representative isolates were distributed to 7 multi-membered (42 isolates) and 9 single membered groups. None of the isolates from these multi-membered groups were considered further as they corresponded with 7 out of the 37 multi-membered taxa that encompassed strains isolated using the DDC procedure. Similarly, 7 out of the 9 single membered clusters were equated with colour groups containing isolates from the DDC method. The largest colour group encompassed 94 isolates which formed a grayish aerial spore mass, which later turned black, a grayish-yellow reverse colour and a yellow diffusible pigment on oatmeal agar, but did not pro-

duce melanin pigments on peptone-yeast extract-iron agar. Apart from the production of the yellow diffusible pigment these properties are consistent with the assignment of the colour group 3 isolates to the S. violaceusniger clade (Sembiring et al. 2000). Curie-point pyrolysis mass spectrometry Excellent agreement was found between the results of the triplicate analyses of each strain, that is, the members of each triplicate set were either superimposed or occupied adjacent positions on the ordination plot (data not shown). Similarly, 4 out of the 5 duplicated cultures grouped adjacent to one another. All of the isolates were recovered in a single pyrogroup (data not

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Figure 1. Neighbour-joining tree (Saitou & Nei 1987) showing relationships between the representative isolates and marker strains belonging to the Streptomyces violaceusniger clade based on almost complete 16S rDNA sequences. The asterisks denote branches that were also recovered using the least-squares (Fitch & Margoliash 1967) and maximum likelihood (Felsenstein 1981) treeing algorithms. The numbers at the nodes indicate the level of bootstrap support (%) based on a neighbour-joining analysis of 1000 resampled datasets. The arrow indicates the estimated root position of the tree.

359 shown). Twelve isolates drawn from this pyrogroup were chosen for the characterisation studies. Phylogenetic relationships The assignment of the 12 representative colour group 3 isolates to the S. violaceusniger clade of the 16S rDNA streptomycete tree is supported by the results from all three treeing algorithms. The taxonomic integrity of the clade was also supported by a relatively high bootstrap value (63%) in the neighbour-joining analysis (Figure 1). It is also evident from this gure that all of the isolates formed a subclade which included the type strain of S. griseiniger. The subclade was recovered in all of the analyses and was supported by a bootstrap value of 99% in the neighbour-joining analysis. It is evident from the 16S rDNA similarity values shown in Table 3 that isolates A10P1, A14P1, A4R2, B22P3, C4R3 and D13P3 are well separated both from one another and from the type strain of S. griseiniger. The closest 16S rDNA similarity value found between these strains was between S. griseiniger NRRL B1865 and isolate D13P3; the shared similarity value found between these strains, 99.2%, corresponds to 11 nucleotide (nt) differences at 1482 nt sites. The 16S rDNA similarity values and the associated nucleotide differences found between the remaining isolates and the most closely related marker strains are shown in Table 3. Accession numbers The nucleotide sequence accession numbers for the tested organisms are as follows: A10P1 (AJ 391834), A14P1 (AJ 391830), A33R1 (AJ 391832), A4R2 (AJ 391835), A23R2 (AJ 391829), B13P3 (AJ 391836), B22P3 (AJ 391833), B23P1 (AJ 391825), C4R3 (AJ 391827), C9P3 (AJ 391828), D13P3 (AJ 391831) and D6R1 (AJ 391826). Chemical, cultural and morphological properties All of the isolates produced whole-organism hydrolysates rich in LL-A2pm and formed rugose ornamented spores in spiral spore chains, as exemplied in Figure 2. It is evident from the dendrogram based on the phenotypic data (Figure 3) that the isolates form an heterogeneous assemblage, the constituent members of which can be readily distinguished from the marker strains representing the validly described species classied in the S. violaceusniger clade. Isolates A10P1, A14P1, A4R2, B22P3, C4R3 and D13P3 can be distinguished from one another and from the type strains of species assigned to the S. violaceusniger clade using a combination of phenotypic properties (Table 4).

Discussion Large numbers of putatively novel streptomycetes were isolated from the environmental samples collected from in and around the root system of Paraserianthes falcataria. The modied DDC procedure was more effective in extracting streptomycete propagules from the environmental samples than the tumble-shaking technique. This result is in line with those from previous studies which showed that the DDC procedure was more effective in extracting actinomycete propagules from soil than classical shaking techniques (Mano 1995; Atalan et al. 2000). There was also evidence that members of some colour groups were associated with specic fractions derived from the application of the DDC procedure. A similar observation was made with respect to members of three putatively novel streptomycete species isolated from rhizosphere soil (Atalan et al. 2000). These observations suggest that associations between taxonomically different streptomycetes and the particulate components of soil may be major factors in limiting representative sampling of streptomycete communities in natural habitats. The multistage DDC procedure, which involves the use of mild detergent (sodium cholate), buffering (Tris buffer), attenuated physical disruption (mild ultrasonication) and ionic shock (distilled water), may be effective in breaking down such associations. The presence of large populations of streptomycetes in the rhizosphere of P. falcataria is consistent with previous reports which showed that actinomycetes are common in plant root systems (Watson & Williams 1974; Upton 1994; Atalan et al. 2000), where they may be responsible for the suppression of root-infecting fungi (Williams et al. 1984; TrejoEstrada et al. 1998a, b). Many streptomycetes inhibit fungal growth in vitro (Williams et al. 1983; Crawford et al. 1993; Al-Tai et al. 1999) and some suppress fungal plant diseases in vitro (Rothrock & Gottlieb 1984; Smith et al. 1990; El-Abyad et al. 1993; You et al. 1996). In the present study, it was particularly interesting that large numbers of isolates were provisionally assigned to the Streptomyces violaceusniger clade (Sembiring et al. 2000), as members of this taxon in-

360
Table 3. 16S rDNA similarity values (%) and number of nucleotide differences between some of the representative isolates and the type strains of the most closely related species classied in the Streptomyces violaceusniger clade

Figure 2. Scanning electron micrograph showing the spore ornamentation of Streptomyces strain A4R2.

361

Figure 3. A dendrogram showing relationships between the representative isolates and marker strains belonging to the Streptomyces violaceusniger clade based on the Ssm/UPGMA analysis. Table 4. Some phenotypic properties separating the representative isolates from one another and from the type strains of the validly described species assigned to the Streptomyces violaceusniger clade

362 hibit root-infecting fungi both in vivo and in vitro (Rothrock & Gottlieb 1984; Trejo-Estrada et al. 1998a; Al-Tai et al. 1999). In particular, S. violaceusniger strain YCED-9 is antagonistic to the growth of a range of plant pathogenic fungi and suppresses dampingoff disease in lettuce caused by Phythium ultimum (Crawford et al. 1993; Trejo-Estrada et al. 1998b). Streptomycete groups based on the production of a distinct aerial spore mass, colony reverse, and diffusible pigment colours on oatmeal agar and on the formation of melanin pigments on peptone-yeast extractiron agar are predictive as many representatives of such groups key out to previously dened species using computer-assisted identication and PyMS ngerprinting procedures (Goodfellow & Haynes 1984; Williams & Vickers 1988; Atalan et al. 2000). It is, therefore, reasonable to conclude that many different types of streptomycetes are associated with the root system of P. falcataria as the isolates selected from the rafnose-histidine and starch casein isolation plates were assigned to 37 multi-membered and 107 single membered colour groups. It is also encouraging that the members of the largest taxon, colour group 3, formed a homogeneous pyrogroup. In general, good congruence has been found between pyrogroups, dened on the basis of PyMS ngerprinting, and corresponding actinomycete groups delineated using more established taxonomic methods (Sanglier et al. 1992; Goodfellow et al. 1996, 1998; Ferguson et al. 1997; Trujillo & Goodfellow, 1997; Colquhoun et al. 2000). The representatives of colour group 3 have properties characteristic of members of the S. violaceusniger clade, notably their ability to form spiral chains of rugose ornamented spores. Seven species have been assigned to this clade, S. albiaviniger Sembiring et al. 2000, S. geldanamycinus Sembiring et al. 2000, S. griseiniger Sembiring et al. 2000, S. hygroscopicus (Jensen 1931) emended Sembiring et al. 2000, S. malaysiensis Al-Tai et al. 1999, S. melanosporofaciens (Arcamone et al. 1959) emended Sembiring et al. 2000 and S. violaceusniger (Waksman & Curtis 1916) emended Sembiring et al. 2000. The most closely related type strains, S. geldanamycinus NRRL 3602T and S. melanosporofaciens NRRL B-12234T , share a 16S rDNA sequence similarity of 99.2%, which corresponds to 7 nucleotide differences, and a DNA:DNA relatedness value of 66%, and can be readily distinguished both from one another and from representatives of the remaining species using a battery of phenotypic properties (Labeda & Lyons, 1991; Sembiring et al. 2000). The minimum level of DNA relatedness recommended for the assignment of strains to a genomic species is 70% (Wayne et al. 1987) though extensive studies on streptomycetes indicate that homology values above 80% correspond to species level relatedness in this genus (Labeda 1993, 1998; Labeda & Lyons 1992). Minimal standards for the denition of Streptomyces species, such as those classied in the S. violaceusniger clade, should be based on a combination of genotypic and phenotypic data (Mano et al. 1995; Kim, S.B. et al. 1998; Kim, B. et al. 1999, 2000). In the present study, isolates A10P1, A14P1, A4R2, B22P3, C4R3 and D13P3 from colour group 3 were distinguished both from one another and from representatives of the validly described species classied in the S. violaceusniger clade by between 11 and 36 16S rDNA nucleotide differences and by a number of phenotypic properties (Table 4). Nucleotide differences within this range have been reported for several validly described species belonging to the S. violaceusniger clade (Sembiring 2000; Sembiring et al. 2000). It is, therefore, concluded from the genotypic and phenotypic data that the six isolates be given species status in the genus Streptomyces.

Description of Streptomyces rhizosphaerius sp. nov. (rhi.zo.sphaeri.us. Gr.n. rhiza, root; Gr. n. sphaera, sphere; N.L. masc. adj. rhizosphaerius belonging to the sphere of the root). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The strain degrades adenine and pectin and grows at 10 C; other phenotypic properties are shown in Table 4. The strain was isolated from the rhizosphere of the tropical legume, Paraserianthes falcataria. The type strain is A10P1 (= DSM 41760 = NCIMB 13674).

Description of Streptomyces asiaticus sp. nov. (a. si. a ti. cus. L. adj. asiaticus Asian). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The strain degrades pectin and grows at 45 C; other

363 phenotypic properties are shown in Table 4. The strain was isolated from the rhizosphere of the tropical legume, Paraserianthes falcataria. The type strain is A14P1 (= DSM 41761= NCIMB 13675). non-rhizosphere soil adjacent to a stand of the tropical legume, Paraserianthes falcataria. The type strain is C4R3 (= DSM 41766 = NCIMB 13681).

Description of Streptomyces cangkringensis sp. nov. Description of Streptomyces indonesiensis sp. nov. (in. do. ne. si. ensis. N.L. adj. indonesiensis pertaining to Indonesia). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The strain degrades pectin but not adenine; other phenotypic properties are shown in Table 4. The strain was isolated from the rhizosphere of the tropical legume, Paraserianthes falcataria. The type strain is A4R2 (= DSM 41759 = NCIMB 13673). (cang. krin. gensis. N.L. adj. cangkringensis pertaining to Cangkringan, a place in Java, Indonesia). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The organism degrades pectin but not adenine and grows at 45 C; other phenotypic properties are shown in Table 4. The strain was isolated from non-rhizosphere soil adjacent to a stand of the tropical legume, Paraserianthes falcataria. The type strain is D13P3 (= DSM 41769 = NCIMB 13684). The present study provides further evidence that streptomycetes which produce spiral chains of rugose ornamented spores form a distinct 16S rDNA clade within the evolutionary radiation occupied by the genus Streptomyces. However, additional comparative studies are needed to resolve the taxonomic status of the remaining representative strains of colour group 3 though the 16S rDNA sequence data suggest that isolates A33R1 (DSM 41763 = NCIMB 13677), B23P1 (DSM 41765 = NCIMB 13680) and D6R1 (DSM 41768 = NCIMB 13683) may belong to a single species. It also seems likely that such studies will lead to the circumscription of additional species within the S. violaceusniger clade. Further studies are also needed to establish the roles that the constituent members of this clade play in the soil ecosystem, notably their interactions with fungal pathogens in root systems. Molecular ecological surveys show that members of many microbial taxa have still to be isolated in pure culture (Embley & Stackebrandt 1997; Bull et al. 2000). Ribosomal RNA gene studies, in particular, have extended the boundaries of microbial diversity though exclusive reliance on this approach may distort views on species richness in natural habitats. Thus, Li et al. (1999) recognised very few cloned 16S rDNA actinomycete sequences in deep sea sediments known to contain members of a range of actinomycete genera (Colquhoun et al. 1998a, b). It is apparent from

Description of Streptomyces javensis sp. nov. (ja. vensis. N.L. adj. javensis pertaining to Java, Indonesia). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The organism degrades xylan and does not grow at 45 C; other phenotypic properties are shown in Table 4. The strain was isolated from non-rhizosphere soil adjacent to a stand of the tropical legume, Paraserianthes falcataria. The type strain is B22P3 (= DSM 41764 = NCIMB 13679).

Description of Streptomyces yogyakartensis sp. nov. (yog. ya. kar. ten sis. N.L. adj. yogyakartensis pertaining to Yogyakarta, Indonesia). Spore chains are Spirales; the spore surface is rugose. On oatmeal agar the spore mass is gray, the substrate mycelium grayish-yellow and the diffusible pigment yellow. Melanin pigments are not produced. The organism grows at 45 C, degrades adenine but does not reduce nitrate; other phenotypic properties are shown in Table 4. The strain was isolated from

364 the present study that selective isolation and characterisation of streptomycete communities can be used to complement data obtained using conventional molecular ecological methods and that considerable taxonomic diversity is encompassed within established groups of streptomycetes. The isolation of representative members of complex streptomycete communities also has a signicant advantage over conventional molecular approaches as representative isolates can be readily screened for novel bioactive compounds and tested for their potential as biocontrol agents.
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Acknowledgements The work described in this paper was carried out within the UK-Indonesian Biodiversity for Biotechnology Development Project (19941999) funded by the UK Department for International Development (DFID). The authors would like to thank the British Council and the International Institute for Biotechnology (wwwbio.ukc.ac.uk/IIBMIRCEN/) for facilitating the collaboration established during this project. The authors are also indebted to Professor H.G. Trper for help with the nomenclature of the new species.

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