FEMS Microbiology Ecology 42 (2002) 419^429

www.fems-microbiology.org

New PCR primers for the selective ampli¢cation of 16S rDNA from
di¡erent groups of actinomycetes1
Paolo Monciardini, Margherita Sosio, Linda Cavaletti, Claudia Chiocchini 2 ,
Stefano Donadio 
Biosearch Italia, via R. Lepetit 34, 21040 Gerenzano (VA), Italy

Received 28 May 2002; received in revised form 2 August 2002; accepted 4 August 2002

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First published online 4 September 2002

Abstract

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive
metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing
unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA
from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from
the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good
sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific
amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to
environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed
to new groups of actinomycetes.
6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Polymerase chain reaction primer ; Environmental clone; Soil DNA; Actinomycetales

1. Introduction tosporangiaceae and Thermomonosporaceae also produce

Actinomycetes are Gram-positive bacteria characterized
by high G+C content in their DNA. They can be found in
a wide range of habitats, and are particularly abundant in
soil [1]. They also produce several structurally diverse bio-
active compounds of pharmaceutical and agricultural im-
portance. Although the order Actinomycetales encom-
passes more than 80 di¡erent genera [2], the majority of
bioactive metabolites have been isolated from Streptomy-
ces species. Nevertheless, microorganisms belonging to the
families Micromonosporaceae, Pseudonocardiaceae, Strep-
* Corresponding author. Tel. : +39 (0) 96474243;
Fax : +39 (0) 96474238.
E-mail address : sdonadio@biosearch.it (S. Donadio).
1
This work is dedicated to the memory of Prof. Franco Tato', former
president of the Italian Society for General Microbiology and Microbial
Biotechnologies.
2
Present address: Biochemie-Fachberech Chemie, Philipps-University
of Marburg, Hans-Meerwein Str., D-35032 Marburg, Germany.
0168-6496 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 3 5 3 - 7
valuable compounds, including erythromycin, gentamicin,
rifamycin, teicoplanin and vancomycin [3,4]. It is not cur-
rently known whether the higher number of streptomycete
metabolites re£ects an increased capacity to produce sec-
ondary metabolites, a larger strain diversity, or if it simply
depends on a higher number of strains screened for bio-
active metabolites. This latter possibility may re£ect their
ease of isolation in comparison to many other actinomy-
cete genera.
Searching for new microbial metabolites requires strat-
egies directed at decreasing the probability of identifying
known compounds. One approach involves the screening
of metabolites produced by actinomycete groups less ex-
ploited than Streptomyces in past screening e¡orts [5,6].
Because they are frequently outcompeted by faster grow-
ing strains, the application of selective methods is a pre-
requisite for the successful isolation of the less common
actinomycete genera [7]. However, the inability to isolate a
particular genus from a given source could depend either
on the absence of that genus, or on the use of inadequate
methods. Knowing a priori the microbial population

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420 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429

present in a source could allow the utilization of proce-
dures appropriate for the isolation of desired genera.
The application of molecular techniques to environmen-
tal samples has allowed the analysis of microbial diversity
without the need for cultivation [8], showing that tradi-
tional cultivation-based techniques greatly underestimate
the microbial diversity present in environmental samples
[8,9]. For this type of analysis, one of the most widely used
markers is 16S rDNA, whose sequence also allows bacte-
rial classi¢cation and the reconstruction of phylogenetic
relationships [10]. Taking advantage of the presence in
the 16S rDNA of small regions diagnostic of phylogeneti-
cally correlated groups, speci¢c polymerase chain reaction
(PCR) primers have been designed for the Actinomycetales
genera Pseudonocardia, Saccharomonospora and Saccharo-
polyspora [11,12], as well as for the analysis of community
structure by denaturing gradient gel electrophoresis using
2. Materials and methods
2.1. Bacterial strains and DNAs
Bacterial strains used in this study were obtained from
the Biosearch Italia collection and are listed in Table 1. All
actinomycete strains were grown in AF/MS medium (per
liter : 20 g glucose, 2 g yeast extract, 8 g soybean £our, 1 g
NaCl, 4 g CaCO3 ) at 28‡C for 6 days. Escherichia coli and
Bacillus subtilis were grown in Luria Broth at 37‡C for
16 h. Genomic DNA was extracted from bacteria with
the PUREGENE1 DNA isolation kit (Gentra Systems)
according to manufacturers instructions and quanti¢ed
with the PicoGreen0 dsDNA Quantitation kit (Molecular
Probes). Clostridium perfringens DNA was purchased from
Sigma.

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actinomycete-speci¢c primers [13].
The objective of the present work was the development
of molecular methods for assessing microbial community
composition with respect to ¢lamentous actinomycetes. In
this paper we describe six new primer sets for the speci¢c
ampli¢cation of 16S rDNA from di¡erent groups of acti-
nomycetes.
2.2. Primer design
A total of 423 16S rDNA sequences of di¡erent groups
of Actinomycetales were retrieved from the RDP-II Data-
base Release 8.0 [14] and aligned using the program PILE-
UP of the Wisconsin Package, Version 9.1 (Genetics Com-
puter Group), following a multi-step approach. First,

Table 1
Bacterial strains and ampli¢cation results
Straina PCR resultsb
BAC ACT STP STM MMS TMM DCT
Streptomyces coelicolor ATCC 10147 + + ^ + ^ ^ ^
Streptomyces lividans ATCC 19844 + + ^ + ^ ^ ^
Streptomyces cinnamoneus NRRL B-1285 + + ^ + ^ ^ ^
Streptomyces avermitilis ATCC 31267 + + ^ + ^ ^ ^
Streptomyces glaucescens DSM 40716 + + ^ + ^ ^ ^
Micromonospora carbonacea ATCC 27114 + + ^ ^ + ^ ^
Actinoplanes teichomyceticus ATCC 31121 + + ^ ^ + ^ ^
Actinoplanes ianthinogenes ATCC 21884 + + ^ ^ + ^ ^
Dactylosporangium matsuzakiense ATCC 31570 + + ^ ^ + ^ +
Dactylosporangium fulvum ATCC 43301 + + ^ ^ + ^ +
Dactylosporangium sp. ID 83892 + + ^ ^ + ^ +
Kibdelosporangium aridum ATCC 39323 + + ^ ^ ^ ^ ^
Saccharomonospora viridis ATCC 15345 + + ^ ^ ^ ^ ^
Saccharotrix australiensis ATCC 31497 + + ^ ^ ^ ^ ^
Amycolatopsis mediterranei ATCC 13685 + + ^ ^ ^ ^ ^
Planobispora rosea ATCC 53773 + + + ^ ^ ^ ^
Planomonospora sp. ID 77572 + + + ^ ^ ^ ^
Nonomuraea sp. ATCC 39727 + + + ^ ^ ^ ^
Streptosporangium cinnabarinum DSM 44094 + + + ^ ^ ^ ^
Streptosporangium sp. ID 71232 + + + ^ ^ ^ ^
Microbispora sp. ID 75340 + + + ^ ^ ^ ^
Actinomadura madurae DSM 43067 + + ^ ^ ^ + ^
Actinomadura macra ATCC 31286 + + ^ ^ ^ + ^
Actinomadura libanotica ATCC 35576 + + ^ ^ ^ + ^
B. subtilis ATCC 8185 + ^ ^ ^ ^ ^ ^
E. coli ATCC 10536 + ^ ^ ^ ^ ^ ^
C. perfringens + ^ ^ ^ ^ ^ ^
a
Codes for strain collections are: ATCC, American Type Culture Collection (Rockville, MD, USA); DSM, Deutsche Sammlung Von Mikroorganismen
und Zellkulturen GMBH (Braunshweig, Germany); NRRL, Ars Patent Culture Collection (Peoria, IL, USA); ID, Biosearch Italia.
b
Ampli¢cations using 5^10 ng genomic DNA and the primers of Table 2. Symbols + and ^ refer to the presence and absence, respectively, of a product
of the expected size.

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 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429 421

Table 2
PCR primers
Primer Sequence Positiona Reference
F27 5P-AGAGTTTGATCMTGGCTCAG-3P 8^27 [13]
R1492b 5P-TACGGYTACCTTGTTACGACTT-3P 1492^1513 [13]
243F 5P-GGATGAGCCCGCGGCCTA-3P 226^243 [13]
A3Rb 5P-CCAGCCCCACCTTCGAC-3P 1414^1430c This study
21F 5P-GACGAARNTGACGTGTA-3P 407^424c This study
959Rb 5P-CGTTGCGTCGAATTAAGCAA-3P 952^971 This study
M2F 5P-SAGAAGAAGCGCCGGCC-3P 492^508 This study
Sm6F 5P-GGTGGCGAAGGCGGA-3P 721^735 This study
Sm5Rb 5P-GAACTGAGACCGGCTTTTTGA-3P 1283^1303 This study
T3F 5P-GGGAGAATGGAATTCCC-3P 665^681 This study
T8Rb 5P-CCCCACCTTCGACC-3P 1413^1426c This study
D3F 5P-GCGGCTTGTTGCGTCAG-3P 585^601 This study
D2Rb 5P-CCGCTGGCAACATCGAACA-3P 1116^1134 This study
a
Numbering refers to the 16S sequence of E. coli rrnB (GenBank J01695, [29]), unless otherwise indicated.
b
Primer anneals to the complementary strand.
c
Numbering refers to the 16S sequence of Streptosporangium vulgare DSM 43802T (GenBank X89955, [30]).

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sequences were aligned within each genus to obtain genus-
speci¢c consensi. These consensi were then aligned within
families, giving a family level consensus for Micromono-
sporaceae, Streptomycetaceae, Streptosporangiaceae and
Thermomonosporaceae. The family level consensi were
aligned with each other and with consensi obtained in
the same way for each of the suborders Actinomycineae,
Corynebacterinae, Frankineae, Micrococcineae, Propioni-
bacterinae and Pseudonocardineae. Aligned sequences
were then visually compared for the identi¢cation of re-
gions showing a high degree of conservation within the
selected family, but having at their 3P-end one or more
mismatches with the consensi of other groups. Possible
primers were evaluated by probing their sequences against
the whole RDP-II database through the Probe Match soft-
ware (http://rdp.cme.msu.edu/html/index.html), ensuring
that at least 90% of the sequences belonging to the target
groups were recognized by the primer. Non-target groups
of sequences that could potentially yield a non-speci¢c
extension product with one primer were taken into ac-
count when choosing the other primer of the pair. The
Tm of each primer was evaluated through Oligo Calculator
version 3.01 (http://www.basic.nwu.edu/biotools/oligo-
calc.html) and primer lengths were adjusted in order to
minimize Tm di¡erences within each primer pair. The
primers used are listed in Table 2.
2.3. PCR ampli¢cations
All ampli¢cation reactions were performed in a ¢nal
volume of 50 Wl, containing 50 mM KCl, 10 mM Tris^
HCl, pH 8.3, each primer at 500 nM, each dNTP at
0.2 mM, 1.5^3.5 mM MgCl2 , 1.5^2.5 U of AmpliTaq or
AmpliTaq Gold DNA Polymerase (Applied Biosystems)
and 3 Wl of 10UDenhardt’s reagent (per liter : 2 g Ficoll
400, 2 g polyvinylpyrrolidone, 2 g bovine serum albumin)
[15]. Optimized cycling parameters, enzyme and MgCl2
concentration for each primer pair are shown in Table 3.
Each ampli¢cation program was initiated by denaturation
for 5 min at 95‡C (10 min when using AmpliTaq Gold)
and ¢nished with an extension step of 10 min at 72‡C.
Reactions were performed either in a PE 9600 (Applied
Biosystems) or in a PCRExpress thermal cycler (Hybaid
Ltd.) equipped with a gradient block for optimization of

Table 3
PCR conditions
Target Code Primers Ampli¢ed Enzyme unitsa MgCl2 (mM) Cycling parametersb Detection limit
fragment (kb) (pg DNA)
Bacteria BAC F27^R1492 1.5 1.5 1.5 45/94^45/61^120/72 NDc
Actinomycetales ACT 243F^A3R 1.25 1.5 3 45/94^120/68 2
Streptomycetaceae STM Sm6F^Sm5R 0.6 1.5 1.5 45/94^45/65^60/72 91
Micromonosporaceae MMS M2F^A3R 1.0 2.5d 1.5 45/94^120/68 1
Streptosporangiaceae STP 21F^959R 0.5 1.5 2.5 45/94^45/58^60/72 1^5
Thermomonosporaceae TMM T3F^T8R 0.8 2d 1.5 45/94^45/59^60/72 1
Dactylosporangium DCT D3F^D2R 0.57 1.5 1.5 30/94^90/68 91
a
AmpliTaq DNA Polymerase, except as noted.
b
Numbers before dash refer to time (in s), after dash refer to temperature (in ‡C) for each step in cycle. PCR comprised 30 cycles, except for primers
TMM (35 cycles).
c
ND: Not determined.
d
AmpliTaq Gold DNA Polymerase.

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422 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429
the annealing temperature. As a positive control, all
DNAs yielded the expected ampli¢cation fragment of
about 1.5 kb when tested with the BAC primers (F27
and R1492, Table 2), speci¢c for all bacteria [13].
2.4. Sequencing of PCR products
PCR products were cloned in the pCR0 2.1-TOPO0
vector using the TopoTA cloning0 kit (Invitrogen). The
ligation mixture was used to transform E. coli JM109 us-
ing standard techniques [16]. About 40^200 colonies were
obtained in each cloning experiment. After boiling single
colonies in 40 Wl sterile H2 O for 5 min and removing cell
debris by centrifugation, 5 Wl of the supernatant was used
as template for PCR. PCR products, puri¢ed with the
GFX1 PCR DNA and Gel Band Puri¢cation kit (Amer-
sham Pharmacia Biotech), were directly sequenced with
the BigDye1 Cycle Sequencing kit (Applied Biosystems)
according to manufacturer’s instructions, and run on an
ABI Prism 310 automatic sequencer (Applied Biosystems).
All sequences were read once with one primer (the forward
primer utilized for the ampli¢cation), for a total length of
400^500 nt.
2.5. Sequence analysis
Sequences were analyzed with the Sequence Match tool
available through the RDP-II web site, which calculates
similarity values (Sab score) with all 16S rRNA sequences
present in the database. All sequences were also inspected
with the program Chimera Check of the RDP-II web site
to test for their possible derivation by PCR-mediated re-
combination of two di¡erent sequences. For the construc-
tion of phylogenetic trees, sequences were aligned with
PILEUP and analyzed with programs included in the
PHYLIP package [17]. Distance matrices were calculated
with DNADIST, using the Maximum Likelihood method
implemented in the program and the method of Jukes and
Cantor [18]. Trees were derived from the distance matrices
using neighbor-joining, UPGMA and the Fitch^Margo-
liash least squares method with contemporary tips (all
methods are included in the PHYLIP package). All anal-
yses were performed on a bootstrapped data set contain-
ing 100 replicates (generated by the program SEQBOOT).
2.6. DNA extraction from soil samples
Soil A (from a temperate forest near Siena, Italy) and
soil B (from an unspeci¢ed location in Niger) were ran-
domly chosen from the Natural Source Collection of Bio-
search Italia, which contains soils collected since the mid
1970s. Soil C was collected from a temperate forest in
Gerenzano, Italy, in August, 1999. After collection, all
soils were air-dried at 30‡C for 2 days and stored in a
sealed container at room temperature (this is a common
procedure for the isolation of sporogenic actinomycetes).
FEMSEC 1414 7-11-02
DNA was extracted from 250 mg soil using the Ultra
Clean1 Soil DNA Isolation kit (MoBio Laboratories
Inc.), and resuspended in 50 Wl of the elution solution
provided. Soil DNA was quanti¢ed with the PicoGreen0
dsDNA Quantitation kit.
3. Results
3.1. Development of selective methods
The actinomycete families described by Stackebrandt
and colleagues [2] were used as the starting point for de-
veloping PCR-based methods. PCR primers were designed
and validated in silico as described under Section 2. Even
if primers for all actinomycetes were already available
[13,19], the ampli¢ed fragment did not encompass all the
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taxon-speci¢c primers described here. We thus combined
primer 243F, used for selective ampli¢cation of actinomy-
cete rDNA [13], with primer A3R (Table 2). The resulting
ACT primer set should allow ampli¢cation of a 1.25-kb
fragment, which includes all the positions where the genus-
and family-speci¢c primers are located. While tested on
puri¢ed DNAs from members of a limited number of ac-
tinomycete families (Table 1), the ACT primers ampli¢ed
a wide range of actinomycete DNAs from soil (see below).
However, these primers might not amplify all actinomy-
cete families, since some Corynebacterinae, the family No-
cardiopsaceae and several Micrococcineae are predicted to
be poor targets for the ACT primers.
Serial dilutions of genomic DNA isolated from target
organisms were used to determine the lowest amount of
target DNA yielding a visible PCR product (see Fig. 1).
For all primers, 5 pg DNA or less was su⁄cient to yield a
distinct PCR product in a gel. No ampli¢cation product
was obtained from members of non-target groups (Table
1), with the exception of the STM primers, which yielded a
very faint band from Saccharomonospora viridis, but only
when using 60 ng DNA (Fig. 1a, lane 10). Details of the
PCR protocols and of the sensitivity of each primer pair
are summarized in Table 3.
3.2. Analysis of soil DNA
The primer pairs and ampli¢cation conditions devised
with a limited number of puri¢ed DNAs were further
evaluated against DNA extracted from three soil samples
(A, B and C). Since soil contains a large number of diverse
bacteria, sequence analysis of the cloned PCR products
should allow their phylogenetic classi¢cation, thus estab-
lishing whether they derive from the target groups. Each
primer set yielded a distinct band using 5^10 ng of soil
DNA as template, as exempli¢ed in Fig. 2. For each prim-
er set, the ampli¢cation products obtained from soil DNA
were cloned and a number of clones (between 18 and 30)
partially sequenced. As some sequence di¡erences could
 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429 423

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Fig. 1. Ampli¢cation of genomic DNA. a: STM Primers. Lanes contain PCR products from DNA of: 1, Streptomyces coelicolor ATCC 10147 (10 ng);
2, Streptomyces glaucescens DSM 40716 (10 ng); 3, Streptomyces cinnamoneus NRRL B-1285 (10 ng); 4^9, Streptomyces lividans ATCC 19844 (10 ng,
1 ng, 100 pg, 10 pg, 2 pg, 1 pg, respectively) ; 10, Saccharomonospora viridis ATCC 15345 (60 ng); 11, Actinomadura madurae DSM 43067 (20 ng);
12, Micromonospora carbonacea ATCC 27114 (5 ng); 13, Streptosporangium cinnabarinum DSM 44094 (20 ng); 14, no DNA; X, molecular mass
marker X (Roche Molecular Biochemicals, Mannheim, Germany). b: DCT Primers. Lanes contain PCR products from DNA of: 1, Dactylosporangium
matsuzakiense ATCC 31570 (100 pg); 2^7, Dactylosporangium sp. ID 83892 (5 ng, 500 pg, 50 pg, 5 pg, 1 pg, 0.5 pg, respectively) ; 8, S. lividans ATCC
19844 (10 ng); 9, Sts. cinnabarinum DSM 44094 (20 ng); 10, Actinoplanes teichomyceticus ATCC 31121 (5 ng); 11, M. carbonacea ATCC 27114 (5 ng);
12, Saccharomonospora viridis ATCC 15345 (60 ng); 13, A. madurae DSM 43067 (20 ng); 14, no DNA ; X, molecular mass marker X. PCR conditions
were as described in Table 3.

derive from nucleotide misincorporation, we have conser-

vatively treated as identical those sequences displaying up
to 1% di¡erence. Phylogenetic analyses yielded trees con-
sistent with each other by di¡erent methods. The complete
list of sequences, their accession numbers and the results
of similarity searches are summarized in Table 4. The re-
sults obtained with each primer set are described below.

3.2.1. Actinomycetales
The 1.25-kb products obtained with the ACT primers
from the three soil DNAs were independently cloned and
30 clones partially sequenced. Database searches indicated
that 22 out of the 30 clones correspond to 20 di¡erent
sequences highly similar to 16S rDNA of cultured mem-
bers of the order Actinomycetales (Table 4). Seven other
clones, corresponding to six di¡erent sequences, also clus- Fig. 2. Ampli¢cation products obtained from soil DNA. Lane 1 con-
ter with actinomycetes by phylogenetic analysis (not tains the ampli¢cation product obtained with the TMM primers from
DNA extracted from soil B. Lanes 2^6 contain the ampli¢cation prod-
shown), and most likely derive from uncultured actinomy-
ucts obtained with primers ACT, STM, MMS, DCT and STP, respec-
cetes, as has also been observed by others [19]. The only tively, using DNA from soil C as template. X refers to molecular mass
sequence diverging from Actinomycetales is that of clone marker X (as Fig. 1); sizes of selected fragments (bp) are reported on
ACT-A-4, which is correlated to that of an uncultured the right. PCR conditions were as described in Table 3.

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Table 4
Soil clones
Sequence IDa Acc. No.d Clonese Best match with RDP-II entries Sab scoref
Primersb Soilc No. Acc. No.d Description
ACT A 1 AJ427635 1 Y08536 Pseudonocardia asaccharolytica 0.879
ACT A 2 AJ427636 1 AF005021 Aeromicrobium erythreum 0.921
ACT A 3 AJ427637 1 AB020031 Saccharotrix tangerinus 0.970
ACT A 4 AJ427638 1 X92708 Clone TM226 0.660
ACT A 5 AJ427639 1 AB006160 ‘Sarraceniospora aurea’ 0.905
ACT A 6 AJ427640 1 AF054278 Mycobacterium str. CH-1 0.976
ACT A 7 AJ427641 1 L40617 Frankia sp. str. HR27-14 0.856
ACT B 1 AJ427642 1 AB028653 Planomonospora parontospora 0.987
ACT B 2 AJ427643 2 D87974 Nocardioides sp. str. KP7 0.944
ACT B 3 AJ427644 1 D87974 Nocardioides sp. str. KP7 0.880
ACT B 4 AJ427645 1 X92612 Micromonospora rhodorangea 0.925
ACT B 5 AJ427646 2 X90830 Nocardioides sp. str. EL-17 0.880
ACT B 6 AJ427647 1 AF005020 Kribbella sandramycini 0.923
ACT B 7 AJ427648 1 Z78210 Nocardioides jensenii 0.842
ACT B 8 AJ427649 1 Z78210 Nocardioides jensenii 0.839

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ACT B 9 AJ427650 1 U27857 Clone Nb 0.936
ACT C 1 AJ427651 1 AF016407 Mycobacterium sp. IMVS B76676 0.963
ACT C 2 AJ427652 1 Y07606 Clone DA016 0.784
ACT C 3 AJ427653 1 AF074412 Kitasatospora sp. str. JL 415 0.967
ACT C 4 AJ427654 1 U72718 Frankia sp. Cea 5.1 0.832
ACT C 5 AJ427655 1 Z73391 Clone Ep_T1.240 0.745
ACT C 6 AJ427656 1 Y07606 Clone DA016 0.825
ACT C 7 AJ427657 1 Z73396 Clone Ep_T1.79 0.867
ACT C 8 AJ427658 1 L40617 Frankia sp. str. HR27-14 0.830
ACT C 9 AJ427659 1 Z73396 Clone Ep_T1.79 0.774
ACT C 10 AJ427660 1 AJ005005 Mycobacterium shimoidei 0.901
ACT C 11 AJ427661 2 Z73385 Clone Ep_T1.12 0.803
STM A 1 AJ427676 1 D63868 Streptomyces bottropensis 0.951
STM A 2 AJ427677 1 Z76683 Streptomyces albido£avus 1.000
STM A 3 AJ427678 1 D63865 Streptomyces acidiscabies 0.964
STM A 4 AJ427679 1 U94490 Streptomyces thermocaboxydus 0.903
STM A 5 AJ427680 1 Z76683 Streptomyces albido£avus 0.942
STM A 6 AJ427681 1 Y15502 Streptomyces griseus 0.990
STM A 7 AJ427682 1 X87320 Streptomycetaceae str. SR70 0.946
STM A 8 AJ427683 2 AJ007425 Streptomyces sp. str. CFBP4508 0.938
STM A 9 AJ427684 1 AB018092 Streptomyces armeniacus 0.886
STM A 10 AJ427685 1 D63868 Streptomyces bottropensis 0.957
STM A 11 AJ427686 1 AF186411 Clone UC32f 0.774
STM C 1 AJ427668 1 AB024442 Streptomyces kasugaensis 0.928
STM C 2 AJ427669 1 X99943 Streptomyces griseocarneus 0.867
STM C 3 AJ427670 1 X81573 Amycolatopsis orientalis subsp. lurida 0.945
STM C 4 AJ427671 1 AB024443 Streptomyces albulus 0.913
STM C 5 AJ427672 3 AF101415 Streptomyces sp. CN 732 0.926
STM C 6 AJ427673 1 AB024440 Streptomyces albulus 0.935
STM C 7 AJ427674 2 AF101415 Streptomyces sp. CN 732 0.893
STM C 8 AJ427675 1 Y15502 Streptomyces griseus 0.990
STP C 1 AJ427662 3 X97892 Nonomuraea salmonea 0.967
STP C 2 AJ427663 1 X97892 Nonomuraea salmonea 0.965
STP C 3 AJ427664 1 AB018046 Microbispora corallina 0.977
STP C 4 AJ427665 3 D85496 Planotetraspora mira 0.959
STP C 5 AJ427666 2 D85496 Planotetraspora mira 0.971
STP C 6 AJ427667 12 X89955 Streptosporangium vulgare 1.000
MMS A 1 AJ427703 1 U58525 Actinoplanes philippinensis 0.942
MMS A 2 AJ427704 2 X93199 Catellatospora ferruginea 0.955
MMS A 3 AJ427705 1 AB024701 Catenuloplanes crispus 0.939
MMS A 4 AJ427706 1 X92631 Micromonospora rosaria 0.978
MMS A 5 AJ427707 1 X93188 Actinoplanes regularis 0.936
MMS A 6 AJ427708 2 U58525 Actinoplanes philippinensis 0.916
MMS A 7 AJ427709 3 U58526 Actinoplanes auranticolor 0.959
MMS A 8 AJ427710 1 X93202 Catenuloplanes caeruleus 0.935
MMS A 9 AJ427711 1 X93187 Actinoplanes philippinensis 0.918

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Table 4 (Continued).
Sequence IDa Acc. No.d Clonese Best match with RDP-II entries Sab scoref
b c d
Primers Soil No. Acc. No. Description
MMS A 10 AJ427712 1 X92631 Micromonospora rosaria 0.913
MMS B 1 AJ427713 1 X92599 Micromonospora carbonacea 0.970
MMS B 2 AJ427714 1 X93201 Catenuloplanes japonicus 0.979
MMS B 3 AJ427715 2 X92612 Micromonospora rhodorangea 0.954
MMS B 4 AJ427716 2 X92631 Micromonospora rosaria 1.000
MMS C 1 AJ427695 2 X92601 Micromonospora halophytica 0.908
MMS C 2 AJ427696 1 D85479 Couchioplanes caeruleus 0.912
MMS C 3 AJ427697 1 X92631 Micromonospora rosaria 0.929
MMS C 4 AJ427698 1 X93188 Actinoplanes regularis 0.895
MMS C 5 AJ427699 1 X92612 Micromonospora rhodorangea 0.916
MMS C 6 AJ427700 1 X92613 Micromonospora olivasterospora 0.890
MMS C 7 AJ427701 1 X92599 Micromonospora carbonacea 0.948
MMS C 8 AJ427702 1 X92612 Micromonospora rhodorangea 0.933
TMM B 1 AJ427687 2 D85473 Actinocorallia herbida 0.946
TMM B 2 AJ427688 1 X97889 Actinomadura madurae 0.941
TMM B 3 AJ427689 1 AB006155 ‘Streptomycoides glauco£avus’ 0.964

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TMM B 4 AJ427690 2 U49003 Actinomadura cremea 0.926
TMM B 5 AJ427691 6 X89953 Streptosporangium viridialbum 0.826
TMM B 6 AJ427692 2 X89953 Streptosporangium viridialbum 0.806
TMM B 7 AJ427693 2 X89953 Streptosporangium viridialbum 0.822
TMM B 8 AJ427694 2 X89953 Streptosporangium viridialbum 0.808
DCT A 1 AJ427719 7 X93191 Dactylosporangium aurantiacum 0.946
DCT A 2 AJ427720 4 X93196 Dactylosporangium vinaceum 0.946
DCT A 3 AJ427721 1 X93191 Dactylosporangium aurantiacum 0.899
DCT C 1 AJ427717 8 X93196 Dactylosporangium vinaceum 0.956
DCT C 2 AJ427718 2 X93193 Dactylosporangium matsuzakiense 0.960
a
Clone ID. Each clone is denoted by primer set, soil and a progressive number.
b
For primers, refer to Table 2.
c
Soil DNA used for ampli¢cation.
d
EMBL accession number of the corresponding sequence.
e
Number of identical sequences obtained from independent clones from same soil DNA.
f
Sab scores obtained with the Sequence Match program.

member of the class Actinobacteria (clone TM226 [20] ;
Table 4). Phylogenetic analysis (not shown) suggests that
ACT-A-4, like clone TM226, derives from the order Acidi-
microbiales [2]. In summary, 29 of the 30 clones, corre-
sponding to 26 distinct sequences, group with 16S rDNA
of Actinomycetales, indicating a speci¢city of ampli¢cation
of about 97%.
3.2.2. Streptomycetaceae
We sequenced a total of 23 clones obtained from soils A
and C with the STM primers. Of these, 22 (representing 17
di¡erent sequences) are highly related to members of the
genus Streptomyces (Sab values between 0.867 and 1.000;
Table 4). Clone STM-C-3 actually shows as best match
Amycolatopsis orientalis subsp. lurida, but this strain may
be a Streptomyces species [21]. Sequence STM-C-7 may
derive from the recombination of PCR products from
two Streptomyces strains, as suggested by the Chimera
Check program. The a⁄liation of all these sequences to
the family Streptomycetaceae is supported by high boot-
strap values (exceeding 98%) with all treeing methods (not
shown). Only clone STM-A-11 appeared to be unrelated
to Streptomycetaceae. This clone gave the highest match
with sequences from uncultured actinomycetes, although
with low Sab scores (0.774; Table 4), suggesting that STM-
A-11 probably derives from some as yet unclassi¢ed acti-
nobacterium.
3.2.3. Streptosporangiaceae
The STP primers yielded a faint 0.5-kb band from the
three soil samples. Analysis of 22 random clones from soil
C gave highest similarity scores with sequences derived
from genera of the family Streptosporangiaceae, and phy-
logenetic analysis con¢rmed their a⁄liation to the family
(not shown). The cloned PCR products correspond to
three groups. A group of four clones, comprising two dif-
ferent sequences, is highly related to Nonomuraea. The
second group consists of six clones, representing three dis-
tinct sequences related to Planotetraspora or Microbispora
(these two genera have similar 16S sequences in the ana-
lyzed region). The last 12 clones correspond to a single
sequence, related to the 16S rDNA of several Streptospo-
rangium species. It is worth mentioning that the sequenced
region is particularly conserved within the genus Strepto-
sporangium. In fact, the RDP database includes nine
Streptosporangium species that show 97% sequence iden-
tity in a 1450-nt span. However, these same sequences are
identical to STP-C-6 over the considered 450 nt. The se-

FEMSEC 1414 7-11-02

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Fig. 3. Phylogenetic analysis of soil-ampli¢ed Micromonosporaceae. Neighbor-joining tree showing the phylogenetic position of environmental clones ob-
tained from three soil samples with MMS primers. Genetic distances were calculated based on 451 unambiguously aligned positions. Numbers at the
nodes are bootstrap values based on 100 resamplings; only values higher than 60 are shown. Scale bar represents 10 inferred substitutions per 100 nu-
cleotides. The tree was rooted using E. coli 16S rDNA as outgroup. Environmental sequences are indicated as in Table 4, e.g. MMS-C-2 refers to clone
2 from soil C, ampli¢ed with the MMS primers.

quenced region is therefore of little help in appreciating
the diversity of Streptosporangium strains.
3.2.4. Micromonosporaceae
A total of 29 clones, representing at least 22 di¡erent
sequences, was analyzed. Database searches con¢rmed
that all clones were strictly related to members of the
family Micromonosporaceae (Table 4). Fig. 3 shows the
phylogenetic relationships among the cloned sequences
and some 16S sequences from known species. Clones
from the same soil tend to cluster together, but it was
possible to identify similar sequences in di¡erent soils,
such as clone MMS-A-3 and clone MMS-B-2, both related
to members of the genus Catenuloplanes. Bootstrap anal-
ysis con¢rmed the a⁄liation of all soil clones with known
Micromonosporaceae in all 100 resampled data sets. With-
in the family, however, bootstrap values are low for most
nodes, so that only a few clones can be unambiguously
attributed to one genus (e.g. clone MMS-B-1 appears to
derive from a member of the genus Micromonospora).
From this ¢nding, it appears that the sequenced region
(about 450 bp) is of low value for genus di¡erentiation
within Micromonosporaceae.
3.2.5. Thermomonosporaceae
Only soil B gave an ampli¢cation product with the
TMM primers. At least eight di¡erent sequences were ob-
served from 18 clones analyzed. Four sequences, cor-
responding to six clones, showed high similarity to 16S
sequences from Actinocorallia, Actinomadura and ‘Strepto-

FEMSEC 1414 7-11-02

 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429 427

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Fig. 4. Phylogenetic analysis of soil-ampli¢ed Thermomonosporaceae. Neighbor-joining tree showing the phylogenetic placement of soil clones ampli¢ed
with the TMM primers. Genetic distances were calculated based on 696 unambiguously aligned positions. The tree was rooted using E. coli as out-
group. Legend is otherwise as for Fig. 3.

mycoides glauco£avus’ (Table 4). The remaining four se-
quences, representing 12 clones, showed Sab values be-
tween 0.800 and 0.836 with other Thermomonosporaceae
(not shown). Phylogenetic trees consistently divided the
eight di¡erent sequences into two groups: the ¢rst four
sequences strictly associated with known Thermomono-
sporaceae, while the remaining four constituted a separate
clade. In order to gather more information about this
group of clones, the sequence of the entire 0.8-kb segment
was used for database searches. Streptosporangium viridial-
bum was found as the closest relative for all sequences,
albeit with low Sab values (between 0.806 and 0.822; Table
4). From a phylogenetic analysis based on 696 unambig-
uously aligned positions, this group of clones clusters to-
gether with members of the family Thermomonosporaceae.
Even if they are clearly di¡erent from known genera, these
sequences constitute a monophyletic lineage supported by
a 100% bootstrap value (Fig. 4). For this primer pair too,
therefore, the ampli¢cation can be considered speci¢c,
even if part of the ampli¢ed sequences are divergent
from known Thermomonosporaceae.
3.2.6. Dactylosporangium
The PCR products from soils A and C were cloned.
Sequence di¡erences among the 22 clones analyzed were
low, and altogether four di¡erent sequences were identi¢ed
(Table 4). All clones correspond to Dactylosporangium se-
quences with Sab values between 0.899 and 0.960. The low
variability found in the sequenced clones was expected, as
the sequenced region is highly conserved in most Dactylo-
sporangium strains. Phylogenetic analyses consistently
placed the clones together with Dactylosporangium strains,

FEMSEC 1414 7-11-02

428 P. Monciardini et al. / FEMS Microbiology Ecology 42 (2002) 419^429
with bootstrap values of 100% for all methods. Since the
sequenced region has a good overlap (over 300 nt) with
that obtained with the MMS primers, the sequences
obtained with the two primer sets were compared over
332 unambiguously aligned positions. Dactylosporangium
clones cluster in a single group within the Micromonospo-
raceae, but none of the 23 clones (14 from soil A and nine
from soil C) obtained with the MMS primers is a⁄liated
to this group (Table 4).
4. Discussion
PCR ampli¢cation of 16S sequences has proven to be a
valuable tool for the detection of bacteria in environmen-
tal samples [13,22], and speci¢c PCR primers for some
genera of actinomycetes have been described [11,12,21].
Since the use of partial 16S rDNA sequences in phyloge-
netic studies can be misleading [9,23,24], we tried to design
primers allowing the ampli¢cation of as large a portion of
the 16S gene as possible. Validation of the primer sets with
soil DNA was an important con¢rmation of their speci¢c-
ity, since the primers were screened against a limited num-
ber of puri¢ed DNAs. A rich source such as soil might
have contained good templates originating from non-tar-
get groups of bacteria. However, this was not the case, at
least for the three soils analyzed. Only for two clones did
the ampli¢cation products not correspond to sequences
from target groups. However, both clones showed a low
similarity to known 16S rDNA sequences, suggesting ei-
ther their ampli¢cation from the DNA of uncultured
groups or derivation by the PCR-mediated recombination
of di¡erent 16S RNA gene fragments.
Considering only the four family-speci¢c primers, 54
distinct sequences were obtained from 92 clones examined.
While the existence of di¡erent 16S rRNA sequences with-
in the same strain has been reported [25], it is not currently
known how widespread this phenomenon is within actino-
mycetes. Although this might be the case for some of our
sequences, our data suggest the existence of a large diver-
sity for most of the considered groups of actinomycetes.
Furthermore, even when the soil clones were highly related
to known sequences, they were seldom identical to data-
base entries (only three cases of Sab values of 1.000 were
observed; see Table 4). Since these sequence comparisons
involved a limited region of the 16S rDNA, sometimes of
modest variability within a group, this ¢nding suggests the
existence of new species belonging to known taxa in the
few soils analyzed. In some instances, a few clones clearly
diverged from the 16S rDNA of known genera, even if
they phylogenetically grouped with Actinomycetales or
Thermomonosporaceae. Thus, as yet uncultured taxa may
also be found within well-studied microorganisms such as
the ¢lamentous actinomycetes.
All primers ampli¢ed the expected band from over 50%
of DNAs prepared from various soils (data not shown),
FEMSEC 1414 7-11-02
con¢rming the worldwide distribution of many actinomy-
cete families [26^28]. 1 pg of puri¢ed DNA corresponds to
about 100 genomes, and a typical PCR reaction employed
DNA extracted from approximately 5 mg of soil. Assum-
ing that the e⁄ciency of ampli¢cation remains the same in
the presence of a large excess of non-target DNA, we can
estimate that positive soils contained at least 20 genomes
from Dactylosporangium species per mg. Thus, even a ge-
nus like Dactylosporangium seems to be widely distributed
and relatively abundant in soil.
In conclusion, the primers described here show good
speci¢city and sensitivity. They work well in complex
DNA mixtures extracted directly from soil, and recognize
di¡erent members of the target group, including new se-
quences from uncultured bacteria, giving an accurate in-
sight into microbial diversity in an environmental sample.
These primers can therefore ¢nd a useful application in
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ecological studies, even if we are mainly interested in their
use for actinomycete isolation. One possible application
might be in the prescreening of soils, followed by the re-
trieval of desired strains through the application of selec-
tive isolation procedures. With additional tools in hand, a
better understanding of the distribution and role of ¢la-
mentous actinomycetes in soil microbial communities can
be obtained. In addition, unusual actinomycetes might be
isolated with high frequency, thus exploiting their biosyn-
thetic potential for discovering novel bioactive metabo-
lites.
Acknowledgements
We wish to thank Elena Bossi and Emiliana Corti for
their early contributions to the project, and Elena Maestri
for helpful advice. We are also grateful to all our col-
leagues at Biosearch Italia for stimulating discussions.
This work was partially supported by a grant from the
Italian MURST, legge 451. P.M. and C.C. were supported
by a fellowship from the Italian MURST.
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