Color profile: Disabled

Composite Default screen

1261

Polymerase chain reaction restriction fragment

length polymorphisms for assessing and
increasing biodiversity of Frankia culture
collections1
Erica Lumini and Marco Bosco

Abstract: During the last few years, some Frankia culture collections that maintained a large number of unidentified
and uncharacterized Frankia strains were closed because of funding shortages. To reduce the costs of maintenance, we
evaluated the biodiversity of half of the Frankia strains from our collection, by polymerase chain reaction restriction
fragment length polymorphisms (PCR-RFLPs) of nifDnifK intergenic spacer and 16S23S rDNA intergenic spacer
regions. In this way we were able to reduce the number of strains without reducing the biodiversity of the whole
collection. In general the nifDnifK target proved to be more polymorphic than the rrn target. From 51 isolates of
Elaeagnus frankiae, PCR-RFLP results allowed us to detect 13 identical strains, and to predict that the genomic species
P8 of Akimov and Dobritsa (1992) very likely agrees with genomic species 5 of Fernandez et al. (1989). Moreover, we
revealed genomic groups not yet described, as well as intraspecific variability. For Alnus frankiae, the polymorphisms
shown by both the nif and the rrn PCR-RFLPs revealed three host plant species-specific subgroups inside Frankia alni.
An expandable data base was created to serve as reference for future biodiversity evaluations on both culture
collections and unisolated Frankia populations. It will be accessible by Internet at the International Frankia Website
(http://www.unifi.it/unifi/distam/frankia/international.html).
Key words: Frankia, PCR-RFLP, nifDnifK intergenic spacer, rrn 16S23S intergenic spacer, biodiversity, culture
collections.
Rsum : Ces dernires annes, plusieurs collection de cultures de Frankia contenant un grand nombre de souches non
identifies et non caracterises ont t abandonnes par manque de fonds. Dans le but dobtenir une rduction des
cots de maintien de notre collection, nous avons valu la biodiversit existante dans la moiti des nos souches de
Frankia. En analysant par la mthode raction en chane de la polymrase polymorphismes de la longeur des
fragments de restriction (PCR-RFPLs) les rgions intergniques nifDnifK et rrn 16S23S, nous avons pu reduire le
nombre de souches en culture, sans rduire la biodiversit de la collection. En gnral, ltude de la rgion nifDnifK a
donn plus de polymorphisme que celle de la rgion ribosomale. Entre les 51 Frankia isols dElaeagnus, on a pu
dtecter 13 souches identiques et montrer que les espces gnomiques P8 de Akimov et Dobritsa (1992) et 5 de
Fernandez et al. (1989) pourraient tre une mme espce. En plus, on a dtect des groupes gnomiques pas encore
dcrits et une certaine diversit intra-spcifique. Pour les souches isoles dAlnus, les polymorphismes montrs par les
deux regions gnomiques ont permis de mettre en evidence, chez espce Frankia alni, trois sous-groupes spcifiques
pour chaque espce hte. Pour faciliter les travaux futurs sur la biodiversit des Frankia cultivs ou des populations
non isoles, on a construit une banque de profils PCR-RFLP qui sera accessible via lInternet dans le site Web
International sur Frankia (http://www.unifi.it/unifi/distam/frankia/international.html).
Mots cls : Frankia, PCR-RFLP, nifDnifK IGS, rrn 16S23S IGS, biodiversit, collection de cultures.
Lumini and

Introduction
Bosco 1269
Since the isolation in pure culture of Frankia strain CpI1
(Callaham et al. 1978), several hundred Frankia strains have
been isolated world-wide. Their classification and identification, as well as the safe storage of cultures have been a
big problem for many frankiologists. Thus, Frankia culture

collections generally maintain a large number of uncharacterized and unexploited strains. Problems on isolation and
identification of these actinomycetes have been attributed
to their long generation times (Meesters et al. 1985), to the
requirement of particular isolation factors (Quispel et al.
1989), or to the lack of knowledge of specific nutritional
requirements of strains (Akkermans et al. 1992). The high

Received August 15, 1998.

E. Lumini and M. Bosco.2 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia
Applicata, Universit degli Studi di Firenze, Piazzale delle Cascine 27, 50144 Florence, Italy.
1

This paper was presented at the 11th International Conference on Frankia and Actinorhizal Plants, June 711, 1998, University of
Illinois at UrbanaChampaign.
2
Author to whom all correspondence should be addressed. e-mail: bosco@csma.fi.cnr.it.
Can. J. Bot. 77: 12611269 (1999)

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:07 AM

1999 NRC Canada

Color profile: Disabled

Composite Default screen

1262

variability of isolates in the genus Frankia (Fernandez

et al. 1989; Akimov and Dobritsa 1992; Dobritsa 1998;
Lechevalier 1994; Lumini et al. 1996), and their presently
unknown degree of heterogeneity among worldwide collections, require rapid and reproducible methods to evaluate
their biodiversity. Currently, molecular biological techniques
are available that have the potential both to permit the characterization of large sets of Frankia isolates and to allow
the identification of inoculated Frankia strains directly inside induced nodules (Lumini and Bosco 1996). Polymerase
chain reaction (PCR) based techniques have been used to
characterize unisolated frankiae (Bosco et al. 1994; Simonet
et al. 1994; Rouvier et al. 1996; Nalin et al. 1997). However,
isolation, purification, and phenotypic characterization of
cultured strains are fundamental to the definition of Frankia
species (Dobritsa 1998; Lechevalier and Ruan 1984;
Lalonde et al. 1988; Simon et al. 1989; Mirza et al. 1991)
and for the exploitation of Frankia in forestry.
Unfortunately, during the last few years some important
Frankia culture collections that maintained a large number
of unidentified and uncharacterized strains were closed because of funding shortage. This has caused the loss of some
Frankia biodiversity. In order to keep the costs of maintenance for our evergrowing Frankia collection low, we sought
a way to reduce the number of strains without decreasing
their biodiversity. To date, Frankia biodiversity has been
characterized by means of different DNA-based techniques,
such as restriction fragment length polymorphisms (RFLP;
Dobritsa and Stupar 1989; Nazaret et al. 1991), repeated sequence PCR (REP-PCR; Murry et al. 1995), randomly amplified polymorphic DNA (RAPD; Sellstedt et al. 1992), and
PCR-RFLPs (Jamann et al. 1993; Cournoyer and Normand
1994; Lumini et al. 1996; Rouvier et al. 1996). The last
technique enables one to work either with a small amount of
culture or with field-collected nodules, by targeting specific
bacterial sequences. International bacterial sequence data
bases are presently focused on the ribosomal coding region,
because of its ubiquitous distribution (Woese 1987) and its
high conservation. When working with nitrogen-fixing bacteria, the nitrogenase coding region (nifHDK) is important,
too, as it supports the principal symbiotic function. In the
case of Frankia the nifHDK sequences have already been
used for specific detection and typing of isolated and unisolated strains (Simonet et al. 1991; Cournoyer and Normand
1994; Lumini et al. 1996).
In the present paper, we examined the extent of interspecific and intraspecific variations in both the nifDnifK
intergenic spacer (IGS) and the 16S23S rDNA IGS regions
of 67 Frankia strains, sampled from different host infectivity
groups throughout 12 different culture collections. The approach was based on the PCR-RFLP method, to compare the
patterns of reference Frankia strains and a large set of unidentified ones. The ultimate aim of this work was to create
an expandable data base of PCR-RFLP patterns, and to apply it for (i) detecting redundant entries in culture collections by identifying subcultures of the same Frankia strain,
(ii) identifying strains from different collections that belong
to the same genomic group or species, and (iii) screening
actinorhizal nodules before isolation to both improve the
biodiversity of the collection and check the successful isolation of the actual microsymbionts.

Can. J. Bot. Vol. 77, 1999

Materials and methods

Frankia strain culture and DNA extraction
Frankia strains used in this study and their known characteristics are listed in Table 1. Reference strains are those representing
genomic species previously described (Fernandez et al. 1989;
Akimov and Dobritsa 1992; Lumini et al. 1996). Cultures were
grown at 28C with weekly manual agitation and subcultured every
23 weeks after fragmenting hyphae through a 22-gauge needle
and transfer into K medium (Lumini et al. 1996) for Elaeagnus-infective strains. For Casuarina-infective strains, a defined medium
termed BAP was used (Murry et al. 1984); this was modified for
Alnus-infective strains with the addition of Tween 80 (1 gL1).
Total DNA from Frankia cultures was isolated and purified by
using previously described methods (Simonet et al. 1984). Rapid
protocols to extract total nucleic acids from low amounts of cultured cells (1 mL) or nodule tissues were also used (Lumini and
Bosco 1996).

PCR amplification and restriction enzyme analysis

Amplification of the 16S23S rDNA IGS was performed under
standard conditions with primers FGPS1490-72 (5 TGCGGCTGGATCCCCTCCTT 3) and FGPL132-38 (CCGGGTTTCCCCATTCGG) (Ponsonnet and Nesme 1994).
The intergenic region between nifD and nifK genes was amplified by using oligonucleotide primers FGPD807-85 (5-CACTGCTACCGGTCGATGAA-3) (Jamann et al. 1993) and FGPK333-355
(5 CCGGGCGAAGTGGCT-3) (Nalin et al. 1995), which were designed from conserved regions of the nifD 3 portion and of the
nifK 5 portion, respectively. The nifDnifK IGS seems more appropriate to characterize strains than the nifHnifD IGS, because it
is longer and more variable (Normand et al. 1992; Jamann et al.
1993; Lumini and Bosco 1996; Lumini et al. 1996; Rouvier et al.
1996; Navarro et al. 1997). For uncharacterized strains, i.e., strains
never tested for their host-infectivity range, the V7 hyper variable
region of the 16S ribosomal genes was also analysed using primer
FGPS989ac-79 (5 GGGGTCCGTAAGGGTC 3) or FGPS989e-80
(5 GGGGTCCTTAGGGGCT 3) (Bosco et al. 1992), in association with reverse primer FGPL132-38, to check whether they did
harbor Elaeagnus or Alnus-Casuarina Frankia rrs-signature
(Nazaret et al. 1991).
PCR DNA amplification reactions were carried out in a final
volume of 100 L by the Perkin Elmer GeneAmp PCR System
9600 (Perkin-Elmer Co., Norwalk, Conn.) with Polymed reagents
(Polymed, Florence, Italy), at the following optimized conditions:
reaction buffer (160 mM (NH4)2SO4; 0.1% Tween-20; 67 mM TrisHCl; pH 8.8), 1.5 mM MgCl2, 0.65 M glycerol, 20 M of each
dNTP, 0.1 M of each primer, and 1 U of PolyTaq. Templates consisted of 25 ng of purified DNA from Frankia cultures or one-tenth
volume of single-lobe extracted total nucleic acids. The thermal
profile was as follows: initial denaturation for 3 min at 95C; 35
cycles of denaturation (30 s at 95C), annealing (30 s at 58C), and
extension (30 s at 72C); and final extension at 72C for 3 min. All
experiments were repeated at least twice, always including both
negative (DNA-free) and positive controls. The estimation of amplicon molecular weights was done on both horizontal 1.5% (w/v)
agarose, and 3.5% Metaphor (FMC Bioproduct, Rockland, Maine)
gels in TBE buffer (89 mM Tris borate, 89 mM boric acid, 2 mM
EDTA; pH 8) at 4 Vcm1 for 2 h. The gels were stained in an
aqueous solution containing 0.4 mgL1 of ethidium bromide, then
destained in TBE for 10 min. Gel images were visualized with an
UV transilluminator at 312 nm, captured, and digitized as TIFF
format files by a CCD camera (UVItec Gel Documentation System). The distances that amplicons and DNA ladders migrated
from the wells were automatically measured, and respective molecular weights were calculated by GelCompar 4.0 (Applied Math,
Kortrijk, Belgium).
1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:08 AM

Color profile: Disabled

Composite Default screen

Lumini and Bosco

1263

Table 1. Designations and characteristics of Frankia strains.

No.

Strain*

Original host plant

Genomic species

Geographical origin

Registry no.

Reference

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45

E1
E1A
E3
E4
E6
E13
E15
E15A
E34
E38
E41
E53
E60
Ea112
HR2714
Ea2914
Ea473
Ea26
Ea305
Ea352
Ea49224
Ea49505
Ea74
Ea33
HRX401a
S14
S15
Hr773
Hr611
EAN1pec
K1
K1A
K1e
TtI11
D11
Ea84
G86
G82
EUN1f
CH8
CH12
CH23
CH37
SCN10a
HRN18a

Esp
Ag
Esp
Esp
Esp
Esp
Esp
Ag
Esp
Esp
Esp
Esp
Esp
Ea
Hr
Ea
Ea
Ea
Ea
Ea
Ea
Ea
Ea
Ea
Hr
Sa
Sa
Hr
Hr
Ea
Cos
Ag
Ea
Tt
Ce
Ea
Gsp
Gs
Eu
Hr
Hr
Hr
Hr
Sc
Hr

10
10
10
10
nd
10
10
10
nd
nd
nd
nd
nd
4
4
4
4
4
4
4
4
4
4
4
5
P8
P8
nd
nd
5
nd
nd
nd
nd
nd
nd
nd
nd
6
nd
nd
nd
nd
nd
7

Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Camaldoli, Italy
Ecully, France
Fontcouverte, France
Castelet, France
Battes, France
Miribel, France
Chateauneuf, France
Sutri, Italy
Nolay, France
Nolay, France
Toulon, France
S. Bonnet, France
Ornon, France
Samara, Russia
Samara, Russia
Mont Aiguille, France
Goufond, France
Ohio, U.S.A.
Rio Tercero, Argentina
Florence, Italy
Florence, Italy
Las Vertientes, Chile
Dakar, Senegal
Pont en Royan, France
Singapore
Singapore
Illinois, U.S.A.
Nogent sur Marne, France
Nogent sur Marne, France
Nogent sur Marne, France
Nogent sur Marne, France
Cap, aux oies, Canada
La Bernarde, France

UFI132701
UFI1327101804
UFI132750
UFI132757
UFI132706
UFI1327013
UFI132715
UFI1327151705
UFI132734
UFI132738
UFI132741
UFI132753
UFI132760
ULF130100112
ULF140102714
ULF130102914
ULF130104703
ULF130100206
ULF130103005
ULF130103502
ULF1301049224
ULF1301049505
ULF130100704
ULF130100303
ULF140104001
ANP190106
ANP190106
ULF140107703
ULF140106101
ULQ130100144
ORS060501
UFI0605011703
UFI0605011301
CHC2102011
ORS020602
ULF130100804
nd
ISU0225887
ULQ132500106
ORS14010208
ORS14010212
ORS14010223
ORS14010237
ULQ190201001
ULF140101801

46
47
48
49
50
51
52
53
54
55
56

HrI1
2.1.7
K2
2.1.9
K3, lobe 3
K3, lobe 9
ArI3
Ar25H5
ACoN24d
Ac4
AvcI1

Hr
Hr
Coc
Hr
Cos
Cos
Ar
Ar
Aco
Aco
Ac

11
11
nd
nd
nd
nd
F. alni
F. alni
F. alni
F. alni
F. alni

Firenzuola, Italy
Gullane, Scotland
Argentina
Gullane, Scotland
Carlos Paz, Argentina
Carlos Paz, Argentina
Oregon, U.S.A.
Orleans, France
Orleans, France
Rincine, Italy
Ontario, Canada

UFI140101
UGL140101
nd
UGL140109
(nodule)
(nodule)
HFP013103
ULF013102
ULF01010244
UFI010104
DDB01020110

Bosco et al. 1992

Bosco et al. 1992
Bosco et al. 1992
Bosco et al. 1992
Unpublished
Lumini et al. 1996
Lumini and Bosco 1996
Lumini and Bosco 1996
Unpublished
Unpublished
Lumini et al. 1992
Unpublished
Unpublished
Fernandez et al. 1989
Fernandez et al. 1989
Jamann et al. 1992
Jamann et al. 1992
Fernandez et al. 1989
Jamann et al. 1992
Jamann et al. 1992
Unpublished
Unpublished
Jamann et al. 1992
Fernandez et al. 1989
Normand et al. 1988
Akimov and Dobrista 1992
Akimov and Dobrista 1992
Lumini et al. 1996
Lumini et al. 1996
Lalonde et al. 1981
Gauthier et al. 1984
Unpublished
Unpublished
Carrasco et al. 1992
Diem et al. 1982
Jamann et al. 1992
Unpublished
Savour and Lim 1991
Lalonde et al. 1981
Prin et al. 1991
Prin et al. 1991
Prin et al. 1991
Prin et al. 1991
Mort et al. 1983
Moiroud and FoureReynaud 1983
Lumini et al. 1996
Lumini et al. 1992
This study
Unpublished
This study
This study
Berry and Torrey 1979
Simonet et al. 1989
Simonet et al. 1984
Margheri et al. 1983
Baker et al. 1979

1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:09 AM

Color profile: Disabled

Composite Default screen

1264

Can. J. Bot. Vol. 77, 1999

Table 1. (concluded).
No.
57

Strain*
ACN14a

Original host plant

Ac

Genomic species
F. alni

Geographical origin
Tadoussaq, Canada

Registry no.
ULQ010201401

58
59

AVN17o
ARgP5AG

Av
Arug

2
3

La Toussuire, France
Quebec City, Canada

ULF014101715
ULQ0132105009

60
61
62
63

Ai13
Ai14
Ai15
CeD

Ai
Ai
Ai
Ce

nd
nd
nd
9

Rincine, Italy
Rincine, Italy
Rincine, Italy
Dakar, Senegal

UFI011113
UFI011114
UFI011115
ORS020606

64
65
66
67

M2
BR
Q2
Q6

Ce
Ce
Cc
Ce

9
9
nd
nd

Madagascar
Brazil
Embraha, Brazil
Mahe, Seycelles

ORS020609
ORS020608
UGL0202602
UFI020606

Reference
Normand and Lalonde
1982
Fernandez et al. 1989
Normand and Lalonde
1986
Antonelli et al. 1992
Unpublished
Unpublished
Diem and Dommergues
1983
Nazaret et al. 1989
Mller et al. 1991
Unpublished
Unpublished

*Reference strains are in bold.

Aco, Alnus cordata; Ac, A. crispa; Ag, A. glutinosa; Ai, A. incana; Ar, A. rubra; Arug, A. rugosa; Av, A. viridis; Cc, Casuarina cunninghamiana; Ce,
C. equisetifolia; Coc, Colletia cruciata; Cos, C. spinosissima; Ea, Elaeagnus angustifolia; Esp, E. sp., Eu, E. umbellata; Gsp, Gymnostoma sp.; Gs, G.
sumatrana; Hr, Hippopha rhamnoides; Sa, Shepherdia argentea, Sc, S. canadensis; Tt, Trevoa trinervis.

nd, not determined.

Restriction enzyme analyses of amplicons were performed in a

total volume of 15 L by using 5 U of the respective restriction
endonucleases at the optimal temperature suggested by the manufacturer. The following restriction endonucleases were used: AluI,
CfoI, NciI, MspI, RsaI, NdeII, TaqI, and HaeIII for the nif region
and, CfoI, MspI, RsaI, and HaeIII for the rrn region. The restricted
fragments were resolved by horizontal electrophoresis in TBE
buffer at 15C, by using a 3.5% (w/v) Metaphor gel containing
0.5 g of ethidium bromide per mL. The molecular weight standards used were the 100-bp and 25-bp DNA Ladder (Gibco-BRL)
and were normally placed every 810 lanes, allowing the detection
of restriction fragments shorter than 50 bp. Gels were run at
5 Vcm1 for 35 h. Gel images were captured and digitized as
above, electrophoretic profiles were rescaled and normalized and
entered in a data base for comparison.

Combination and numerical analysis of PCR-RFLP

patterns
All patterns obtained with each of the restriction endonucleases
from the nifDnifK IGS and the 16S23S IGS were assembled to
obtain a single combined restriction pattern for each of the 65
strains listed in Table 3. A similarity matrix between each pair
of combined patterns was calculated using the Dice similarity coefficient, and the genetic distance between each pair of strains
was estimated by using the mathematical model defined by Nei
and Li (1979). A phylogenetic tree was then constructed by using
the FitchMargoliash method with contemporary tips (Fitch and
Margoliash 1967) by using PHYLIP version 3.572c (Felsenstein J.,
University of Washington).

Results
PCR-RFLP analysis of amplified 16S23S rDNA
The 16S23S rDNA amplicons were similar on 1.5% agarose
gel for all 67 Frankia strains examined, but quite different
when molecular weight was estimated on a 3.5% Metaphor
gel. The sizes ranged between 670 bp in AVN17o and
330 bp in Q6. Digestion of the 16S23S IGS with each of
the four restriction enzymes (CfoI, MspI, RsaI, and HaeIII)
produced variable numbers (1621) of fragment patterns,
depending on enzyme discriminating power. The 16S23S

fragment patterns were relatively easy to interpret because

of the simple nature of the patterns (i.e., few restriction
sites). Frankia strains were grouped into the same rrntype when they shared the same combination of restriction
patterns. Twenty four PCR-RFLP rrn-types were recognized
on the basis of restriction patterns (Table 2). The 11 Alnusinfective Frankia strains were divided into six rrn-types.
Among the 51 Elaeagnus frankiae, we could detect 16 different rrn-types. DNA of Frankia strains Q2 and Q6, isolated from Casuarina but never tested for their host
infectivity, amplified with primer FGPS989ac, which is specific for the AlnusCasuarina group, but not with primer
FGPS989e, which is specific for the Elaeagnaceae infectivity group. Moreover, although Frankia strain Q2 always
showed the same profiles that Frankia reference strain CeD,
strain Q6 always showed different patterns, as expected by
its unusually low-sized rrn amplicon.
PCR-RFLP analysis of amplified nifDK IGS
The nifDnifK intergenic region was successfully amplified for 65 strains, yielding fragments of noticeably different
sizes, especially between those belonging to Elaeagnusinfective and Alnus-infective strains. Neverthless, there were
observable differences among strains from the same host
infectivity group. For istance, the nifDnifK IGS in Alnusinfective strains is variable enough in size to discriminate
the strains AVN17o and Ai13 from the strains belonging to
Frankia alni (ArI3, Ar24H5, ACoN24d, Ac4, AvcI1, and
ACN14a). Independent cleavage by the eight enzymes AluI,
CfoI, NciI, MspI, RsaI, NdeII, TaqI, and HaeIII, gave 1522
different restriction patterns, depending on the discriminating power of each enzyme. Frankia strains were grouped
in a nif-type when they shared the same combination of
restriction patterns (Table 3). Fifty Elaeagnus-compatible
Frankia isolates, comprising Frankia strains from Sheperdia,
Gymnostoma, and Trevoa, and the two DNAs obtained from
nodule lobes of Colletia spinosissima were grouped into
22 PCR-RFLP nif-types. Strains belonging to the Alnusinfectivity group were divided into 7 PCR-RFLP nif-types, 3
1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:10 AM

Color profile: Disabled

Composite Default screen

Lumini and Bosco

1265

Table 2. Restriction patterns of amplified 16S23S IGS.

No.*

Strains

Gs

RsaI

MspI

CfoI

HaeIII

rrn types||

113
1430
3133
34
35
36
37
38
39
4043
44
45
4647
48
49
5051
5253
5455
5657
58
59
6062
6366
67

E1
Ea112
K1
TtI11
D11
Ea84
G86
G82
EUN1f
CH8
SCN10a
HRN18a
HrI1
K2
2.1.9
K3 lobe 3
ArI3
ACoN24d
AvcI1
AVN17o
ARGP5AG
Ai13
CeD
Q6

10
45-P8
nd
nd
nd
nd
nd
nd
6
nd
nd
7
11
nd
nd
nd
1
1
1
2
3
nd
9
nd

R1
R2
R2
R2
R2
R3
R4
R5
R3
R6
R7
R3
R3
R8
R9
R10
R11
R11
R12
R13
R14
R15
R12
R16

M1
nr
nr
nr
M2
M3
M4
M5
M6
M7
M8
M9
M10
M10
M11
M12
M13
M13
M14
M15
M16
M17
M18
M19

C1
C2
C2
C2
C3
C4
C5
C6
C7
C5
C8
C9
C10
C11
C12
C13
C14
C14
C15
C16
C17
C18
C19
C20

H1
H2
H3
H4
H5
H6
H7
H8
H9
H8
H9
H10
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21

I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
XX
XXI
XXII
XXIII
XXIV

*Strains with the same PCR-RFLP patterns (see Table 1 for references).

Type strains.

Genomic species. nd, not determined.

nr, no restriction site detected.

||
The letters designate the restriction enzyme used, and the indexed number designates the pattern obtained with this enzyme. The rrn types represent the
combination of patterns obtained with the four enzymes tested on the 16S23S rDNA IGS.

of which overlapped rrn types. All five Casuarina strains

were grouped into a single PCR-RFLP nif-type.
It is worthy that the restriction enzyme NdeII showed the
highest discriminating power for the nifDnifK IGS among
50 Elaeagnus-group strains, giving 20 different restriction
patterns.
Genomic relationships inferred by the combined PCRRFLPs data sets
A PCR-RFLPs database of 395 different bands from 203
different patterns was analysed, and a phylogenetic tree
(Fig. 1) was constructed on the basis of the estimation of genetic distances. It revealed 31 distinct branches, evidencing
the high level of Frankia biodiversity conserved by our collection. Concerning Alnus-infective strains, although the six
Frankia alni strains clustered at 72% similarity level, they
clearly diverged into three subspecific groups. Elaeagnusinfective strains could be divided into three main clusters,
the larger of them being composed by the genomic species 4
and 5 of Fernandez et al. (1989), in accordance with their
high 16SrDNA sequence similarities (Nazaret et al. 1991).
Previously described genomic species were clearly identifiable, with the exception of the genomic species 5 of
Fernandez et al. (1989) and the genomic species P8 of
Akimov and Dobritsa (1992), which were linked on the dendrogram at a 100% similarity level.

Discussion
This work is the first attempt to compare a large set of
cultured Frankia strains by PCR-RFLP analysis of both the
16S23S rDNA IGS and the nifDnifK IGS. We analysed 67
Frankia strains of the three host specificity groups to assess
the biodiversity of Frankia strains in general, rather than to
investigate any particular genomic species or host infectivity
group of strains.
Both targets gave positive signals with all 67 strains, showing
some differences in grouping Frankia strains when amplicons
were analysed by RFLP analysis. In general, the nifDnifK target proved to be more polymorphic than the rrn target, for both
Alnus and Elaeagnus strains. The only exception was represented by the five Casuarina strains, always showing the same
nif-type, but two different rrn-types (Table 2).
For the nif region, the discriminant power of HaeIII was
different if used on Elaeagnus- or on Alnus-infective strains.
Infact, it was the second most discriminating enzyme for
Elaeagnus frankiae (17 patterns), but was a low discriminating one among Alnus frankiae. On the other hand, the
restriction endonuclease AluI had the lowest discriminant
power in Elaeagnus-infective strains (eight patterns), but a
quite high one in Alnus-infective strains (six patterns). These
results, together with the phylogenetic analysis on combined
restriction patterns (Fig. 1), confirmed the hypothesis of
a wide genetic distance between Elaeagnus-infective and
1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:11 AM

Color profile: Disabled

Composite Default screen

1266

Can. J. Bot. Vol. 77, 1999

Table 3. Restriction pattern of amplified nifDnifK fragments.

No.*

Strains

Gs

AluI

113
1417
1820
2122
23
24
2529
30
3133
34
36
37
38
39
4043
44
45
46
47
48
49
5051
No.

E1
Ea112
Ea26
Ea49224
Ea74
Ea33
HRX401a
EAN1pec
K1
TtI11
Ea84
G86
G82
EUN1f
CH8
SCN10a
HRN18a
HrI1
2.1.7
K2
2.1.9
K3 lobe 3
Strains

10
4
4
nd
4
4
5+P8
5
nd
nd
nd
nd
nd
6
nd
nd
7
11
11
nd
nd
nd
Gs

A1
A2
A2
A2
A2
A2
A2
A2
A2
A2
A3
A3
A3
A4
A5
A6
A7
A7
A7
A7
A8
nd
TaqI

5253
54
55
56
57
58
6062
No.

ArI3
ACoN24d
Ac4
AvcI1
ACN14a
AVN17o
Ai13
Strains

1
1
1
1
1
2
nd
Gs

6367

CeD

NciI

MspI

CfoI

Elaeagnus compatible Frankia isolates

Nc1
M1
C1
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc2
M2
C2
Nc3
M3
C3
Nc4
M4
C4
Nc5
M5
C5
Nc6
M6
C6
Nc7
M7
C7
Nc8
M8
C8
Nc9
M9
C9
Nc9
M9
C9
Nc9
M9
C9
Nc9
M9
C10
Nc10
M10
C11
nd
nd
C12
HaeIII
RsaI
NciI
Alnus infectivity group
T1
H18
R16
Nc11
T1
H18
R17
Nc12
T1
H18
R17
Nc13
T2
H19
R18
Nc14
T2
H19
R18
Nc14
T3
H20
R19
Nc15
nd
H21
R20
Nc16
AluI
NciI
MspI
CfoI
Casuarina infectivity group
A15
Nc17
M18
C20

RsaI

HaeIII

NdeII

nif types

R1
R2
R2
R3
R3
R3
R3
R3
R3
R4
R5
R6
R7
R8
R9
R10
R11
R12
R12
R13
R14
R15
AluI

H1
H2
H3
H3
H4
H5
H6
H7
H7
H7
H8
H9
H10
H11
H12
H13
H14
H15
H16
H15
H16
H17
CfoI

N1
N2
N2
N3
N3
N4
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
MspI

I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
XX
XXI
XXII
nif types

A9
A10
A11
A12
A13
nr
A14
RsaI

C13
C14
C15
C16
C17
C18
C19
HaeIII

M11
M12
M13
M14
M15
M16
M17
NdeII

XXIII
XXIV
XXV
XXVI
XXVII
XXVIII
XXIX
nif types

R21

H22

N21

XXX

*Strains with the same PCR-RFLP patterns (see Table 1 for references).

Type strains.

Genomic species. nd, not determined.

The letters designate the restriction enzyme used and the indexed number designates the pattern obtained with this enzyme. The nif types represent the
combination of patterns obtained with the seven enzymes tested on the nifDnifK amplicon.

Alnus-infective strains. Previous data on the high diversity

within Frankia strains belonging to the Elaeagnus-infective
group were also confirmed, as eight new putative genomic
species could be detected among 15 uncharacterized Frankia
strains. However, the 13 Elaeagnus Frankia strains isolated
in Italy always clustered, probably representing an adaptative radiation from a single strain. As an application of
these findings, we are currently successfully using PCR-RFLPs
on new Colletia isolates and their source nodules to verify
that we have indeed isolated the actual Frankia microsymbionts (unpublished data).
Interestingly, the two strains S14 and S15, belonging to
genomic species P8 defined by Akimov and Dobritsa (1992),
formed a unique nif PCR-RFLP cluster (nif-type VII, Table 3; and Fig. 1) together with strains Hr77.3, Hr61.1, and
the type strain HRX401a of the genomic species 5 defined
by Fernandez et al. (1989). As the resolution power of the

target sequence between primer FGPD807-85 and primer

FGPK333-355 is not efficient enough to resolve Frankia
genomic species, we confirmed the above results by analysing the target sequence between primer FGPD807-85 and
primer FGPK700-92 target by MspI, a valuable taxonomical
marker for resolving Frankia genomic species 4, 5, 6, and
10 (Jamann et al. 1992; Lumini et al. 1996). The five cited
strains clustered together and with Frankia EAN1pec, another reference strain of genomic species 5 (data not shown).
This is the first attempt to compare strains belonging to
genomic species described by independent frankiologists.
We believe that the genomic species P8 and the genomic
species 5, independently described by Akimov and Dobritsa
(1992) and Fernandez et al. (1989), could be one and the
same, and we suggest this could be verified by DNADNA
hybridization. This hypothesis is also supported by the phylogenetic tree shown in Fig. 1, and by recent data from
1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:12 AM

Color profile: Disabled

Composite Default screen

Lumini and Bosco

1267

Fig. 1. Genomic relationships among 65 collection Frankia strains, based on the combined nifDnifK IGS and 16S23S IGS PCRRFLPs data set, inferred by using Kitsch and plotted by using Drawgram free software (PHYLIP version 3.572c). *, genomic species
are numbered as described by Fernandez et al. (1989), Akimov and Dobritsa (1992), and Lumini et al. (1996).

Dobritsa (1998), who analysed some of the Frankia strains

included in this work, and showed concordance between
phenotypic clusters and genospecies previously described.
Concerning Frankia strains belonging to the Alnus group,
the polymorphism showed by the 16S23S IGS PCR-RFLPs

confirmed the distance between genomic species 2 and 3,

and Frankia alni (genomic species 1), and gave a new PCRRFLP group (strains Ai13, Ai14, and Ai15). Moreover, we
noticed a subspecific differentiation inside Frankia alni.
When analysed with an appropriate set of enzymes, both tar 1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:12 AM

Color profile: Disabled

Composite Default screen

1268

gets were able to show the same three clusters (Fig. 1). Each
cluster contained two strains from very different geographic
origins (Table 1), but isolated from the same host plant species: Alnus cordata, A. crispa, and A. rubra, respectively.
Such a tendency seems to be present among other Alnus
strains, too. However, the presence of only one strain in
each cluster is insufficient evidence. A larger set of Alnus
spp. nodule DNAs are being analysed to confirm this hypothesis.
The polymorphism showed by the nifDnifK PCR-RFLPs
of Alnus strains confirmed that the nifDnifK intergenic region is quite good to characterize Frankia from Alnus at the
strain level, with the exception of strains ArI3 and Ar24H5.
However, it could resolve clusters at the genomic species
level only after running a phylogenetic analysis of combined
band patterns. Thus, the rapid classification and identification
of Alnus microsymbionts, or new isolates, will need a twotarget protocol: PCR-RFLP of the nifHnifD intergenic region
(Cournoyer and Normand 1994) for genomic species recognition, coupled with PCR-RFLP of the nifDnifK region and
phylogenetical analysis for resolution of similar strains.
Although more sophisticated molecular techniques for
identification of Frankia will be available in the future, the
data presented here and in the other cited works suggest that
we already have a tool that enables us both to make significant analyses of the Frankia biodiversity, and to infer the
genetic structure of the genus Frankia at the species or strain
level. We support the use of PCR-RFLPs to screen biodiversity of worldwide collections and to check nodules
before making the effort to isolate strains. In fact, bulk samples (cultured strains, new isolates, nodules) can be analysed
by this simple and inexpensive method. Large sets of restriction patterns and published nucleotide sequences of the
cor-responding DNA regions could be easily compared by
PHYLIP shared analysis software, for cheap identification
purposes. In order to set up a World Wide Web Frankia
identification system, our PCR-RFLP data base should soon
be linked to the International Frankia Website at the URL:
http://www.unifi.it/unifi/distam/frankia/international.html.

Acknowledgements
We gratefully acknowledge M.C. Margheri, C.T Wheeler,
A. Moiroud, J. Schwencke, S. Dobritsa, and L. Simon for
supplying Frankia strains; L. Oliva for Colletia nodules; and
S. Dobritsa, D. Baker, D. Benson, and P. Normand for useful
discussions. We also thank J. Felsenstein for sharing the
PHYLIP programs; C. Picard for proofreading the French
abstract and commenting on the manuscript; and G., A., and
B. Franceschi for continuous encouragement and interest in
our research. This work was supported in part by grant
96.00625.06 to M.B. from the Italian National Research Council,
by MURST fundings (ex. 40%), and by a Ricerca Scientifica
di Ateneo grant from the Universit degli Studi di Firenze.
E.L. was the recipient of a post-doctoral fellowship from the
Universit degli Studi di Firenze, Florence, Italy.

References
Akimov, V.N., and Dobritsa, S. 1992. Grouping of Frankia strains
on the basis of DNA relatedness. Syst. Appl. Microbiol. 15:
372379.

Can. J. Bot. Vol. 77, 1999

Akkermans, A.D.L., Hahn, D., and Baker, D.D. 1992. The family
Frankiaceae. In The prokaryotes. Edited by A. Balows, H.G.
Trper, M. Dworkin, W. Harder, and K.H. Schleifer. SpringerVerlag, New York. pp. 10691084.
Antonelli, F., Bosco, M., Lumini, E., and Favilli, F. 1992. Survival
of Frankia spp. strains introduced in a disturbed soil. In Abstract of the Sixth International Symposium on Microbial Ecology (ISME 6). University of Barcelona, Barcelona, Spain.
Baker, D., Kidd, G.H., and Dillon, J.T. 1979. Separation of actinomycete nodule endophytes from crushed nodule suspensions by
Sephadex fractionation. Bot. Gaz. 140: 4951.
Berry, A., and Torrey, J.G. 1979. Isolation and characterization in
vivo and in vitro of an actinomycetous endophyte from Alnus
rubra Bong. In Symbiotic nitrogen fixation in the managment of
temperate forests. Edited by J.C. Gordon, C.T. Wheeler, and
D.A. Perry. Forest Research Laboratory, Oregon State University, Corvallis, Oreg. pp. 6983.
Bosco, M., Fernandez, M.P., Simonet, P., Materassi, R., and
Normand, P. 1992. Evidence that some Frankia sp. strains are
able to cross boundaries between Alnus and Elaeagnus host
specificity groups. Appl. Environ. Microbiol. 58: 15691576.
Bosco, M., Jamann, S., Chapelon, C., Simonet, P., and Normand, P.
1994. Frankia microsymbiont in Dryas drummondii nodules is
closely related to the microsymbiont of Coriaria and genetically
distinct from other characterized Frankia strains. In Nitrogen
fixation with non-legumes. Edited by N. A. Hegazi, M. Fayed,
and M. Manib. The American University in Cairo Press, Cairo,
Egypt. pp. 173183.
Callaham, D., Del Tredici, P., and Torrey, J.G. 1978. Isolation and
cultivation in vitro of the actinomycetes causing roots nodulation
in Comptonia. Science (Washington, D.C.), 199: 899902.
Carrasco, A., Schewencke, J., and Caru, M. 1992. Isolation of
Frankia from nodules of Trevoa trinervis: ultrastructural characterization. Can. J. Microbiol. 38: 174180.
Cournoyer, B., and Normand, P. 1994. Characterization of a spontaneous thiostrepton-resistant Frankia alni infective isolate using PCR-RFLP of nif and gln II genes. Soil Biol. Biochem. 26:
553559.
Diem, H.G., and Dommergues, Y.R. 1983. The isolation of Frankia
from nodules of Casuarina. Can J. Bot. 61: 28222828.
Diem, H.G., Gauthier, D., and Dommergues, Y.R. 1982. Isolation
of Frankia from nodules of Casuarina equisetifolia. Can. J.
Microbiol. 28: 526530.
Dobritsa, S. 1998. Grouping of Frankia strains on the basis of susceptibility to antibiotics, pigment production and host specificity. Int. J. Syst. Bacteriol. 48: 12651275.
Dobritsa, S., and Stupar, O.S. 1989. Genetic heterogeneity among
Frankia isolates from root nodules of individual actinorhizal
plants. FEMS Microbiol. Lett. 58: 287292.
Fernandez, M.P., Meugnier, H., Grimont, P.A.D., and Bardin, R.
1989. Deoxyribonucleic acid relatedness among members of the
genus Frankia. Int. J. Syst. Bacteriol. 39: 424429.
Fitch, W.M., and Margoliash, E. 1967. Construction of phylogenetic trees. Science (Washington, D.C.), 155: 279284.
Gauthier, D.L., Frioni, L., Diem, H.G., and Dommergues, Y.R.
1984. The Colletia spinosissimaFrankia symbiosis. Oecol.
Plant. 5: 231239.
Jamann, S., Fernandez, M.P., and Moiroud, A. 1992. Genetic diversity of Elaeagnaceae-infective Frankia strains isolated from various soils. Acta Oecol. 13: 395405.
Jamann, S., Fernandez, M.P., and Normand, P. 1993. Typing
method for N2-fixing bacteria based on PCR-RFLP: application
to the characterization of Frankia strains. Mol. Ecol. 2: 1726.
Lalonde, M., Calvert, H.E., and Pine, S. 1981. Isolation and use of
1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:13 AM

Color profile: Disabled

Composite Default screen

Lumini and Bosco

Frankia strains in actinorhizae formation. In Current perspectives in nitrogen fixation. Edited by A.H. Gibson and W.E Newton. Australian Academy of Sciences, Canberra. pp. 296299.
Lalonde, M., Simon, L., Bousquet J., and Seguin, A. 1988. Advances in the taxonomy of Frankia: Recognition of species alni
and elaeagni and novel subspecies pommerii and vandijkii. In
Nitrogen fixation a hundred years after. Edited by H. Bothe, F.J.
de Brujin, and W.E. Newton. Gustav fisher verlag, Stuttgart.
pp. 671680.
Lechevalier, M.P. 1994. Taxonomy of the genus Frankia (Actinomycetales). Int. J. Syst. Bacteriol. 44: 18.
Lechevalier, M.P., and Ruan, J.S. 1984. Physiology and chemical
diversity of Frankia spp. isolated from nodules of Comptonia
peregrina (l.) Coult. and Ceanothus americanus L. Plant Soil,
78: 1522.
Lumini, E., and Bosco, M. 1996. PCR-restriction fragment length
polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI132715. Appl. Environ. Microbiol. 62: 30263029.
Lumini, E., Bosco, M., Favilli, F., and Wheeler, C.T. 1992. Production of spores in nodules of Alnus glutinosa inoculated with
non-host Frankia strains. Acta Oecol. 23: 506507.
Lumini, E., Bosco, M., and Fernandez, M.P. 1996. PCR-RFLP and
total DNA homology revealed three related genomic species
among broad-host-range Frankia strains. FEMS Microbiol.
Ecol. 21: 303311.
Margheri, M.C., Tredici, M.R., and Florenzano, G. 1983. Isolamento e coltura dellendofita del genere Frankia da noduli radicali di Alnus cordata in Italia. Ann. Microbiol. 33: 137148.
Meesters, T.M., van Genesen, S.T., and Akkermans, A.D.L. 1985.
Growth, acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17
and Cp1.2. Arch. Microbiol. 143: 137142.
Mirza, M.S., Janse, J.D., Hahn, D., and Akkermans, A.D.L. 1991.
Identification of atypical Frankia strains by fatty acid analysis.
FEMS Microbiol. Lett. 83: 9197.
Moiroud, A., and Faure-Reynaud, M. 1983. Influences de quelques
herbicides large spectre sur la croissance et linfectivit de cultures pures de Frankia. Plant Soil, 74: 133136.
Mort, A., Normand, P., and Lalonde, M. 1983. 2-o-Methyl-D-mannose, a key sugar in the taxonomy of Frankia. Can. J. Microbiol.
29: 9931002.
Mller, A., Benoist, P., Diem, H.G., and Schwencke, J. 1991. Agedependent changes in extracellular proteins, aminopeptidase and
proteinase activities in Frankia isolate Br. J. Gen. Microbiol.
137: 27872796.
Murry, M.A., Fontaine, M.S., and Torrey, J.G. 1984. Growth kinetics and nitrogenase induction in Frankia sp. HFPArI3 growth in
batch culture. Plant Soil, 78: 6178.
Murry, M.A., Zhang, D., Schneider, M., and de Bruijn, F.J. 1995.
Use of repetitive sequences and the polymerase chain reaction
(rep-PCR) to fingerprint the genomes of Frankia isolates. Symbiosis, 19: 223240.
Nalin, R., Domenach, A.M., and Normand, P. 1995. Molecular
structure of the Frankia spp. nif D-K intergenic spacer and design
of Frankia genus compatible primer. Mol. Ecol. 4: 483491.
Nalin, R., Normand, P., and Domenach, A.M. 1997. Distribution
and N2-fixing activity of Frankia strains in relation to soil depth.
Physiol. Plant. 99: 732738.
Navarro, E., Nalin, R., Gauthier, D., and Normand, P. 1997. The
nodular microsymbionts of Gymnostoma spp. are Elaeagnus-infective Frankia strains. Appl. Environ. Microbiol. 63: 16101616.

1269
Nazaret, S., Simonet, P., Normand, P., and Bardin, R. 1989. Genetic diversity among Frankia isolated from Casuarina nodules.
Plant Soil, 118: 241247.
Nazaret, S., Cournoyer, B., Normand, P., and Simonet, P. 1991.
Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences. J. Bacteriol.
173: 40724078.
Nei, M., and Li, W.H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl.
Acad. Sci. U.S.A. 76: 52695273.
Normand, P., and Lalonde, M. 1982. Evaluation of Frankia strains
isolated from provenances of two Alnus species. Can. J. Microbiol. 28: 11331142.
Normand, P., and Lalonde, M. 1986. The genetics of actinorhizal
Frankia: a review. Plant Soil, 90: 429453.
Normand, P., Simonet, P., and Bardin, R. 1988. Conservation of nif
sequences in Frankia. Mol. Gen. Genet. 213: 238246.
Normand, P., Gouy, M., Cournoyer, B., and Simonet, P. 1992. Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic interferences. Mol. Biol. Evol. 9: 495506.
Ponsonnet, C., and Nesme, X. 1994. Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal
regions. Arch. Microbiol. 59: 40234030.
Prin, Y., Maggia, L., Picard, B., Diem, H.G., and Goullet, P. 1991.
Electrophoretic comparison of enzymes from 22 single-spore
cultures obtained from Frankia strain ORS140102. FEMS
Microbiol. Lett. 69: 9196.
Quispel, A., Svendsen, A.B., Schripsema, J., Baas, W.J., Erkelens,
C., and Lugtenburg, J. 1989. Identification of a dipterocarpol as
isolation factor for the induction of primary isolation of Frankia
from root nodules of Alnus glutinosa (L.) Gaertner. Mol. PlantMicrobe Interact. 2: 107112.
Rouvier, C., Prin, Y., Reddel, P., Normand, P., and Simonet, P.
1996. Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR
and restriction fragment length polymorphism analysis with
crushed nodules. Appl. Environ. Microbiol. 62: 979985.
Savour, A., and Lim, G. 1991. Characterization of an infective
Frankia (ISU0224887) isolated from nodules of Gymnostoma
sumatranum. Plant Soil, 131: 2127.
Sellstedt, A., Wullings, B., Nystom, U., and Gustafsson, P. 1992.
Identification of Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers. FEMS Microbiol. Lett. 93: 16.
Simon, L., Jabaji-Hare, S., Bousquet, J., and Lalonde, M. 1989.
Confirmation of Frankia species using cellular fatty acids analysis. Syst. Appl. Microbiol. 11: 229235.
Simonet, P., Bosco, M., Chapelon, C., Moiroud, A., and Normand,
P. 1994. Molecular characterization of Frankia microsymbionts
from spore-positive and spore-negative nodules in a natural alder stand. Appl. Environ. Microbiol. 60: 13351341.
Simonet, P., Capellano, A., Navarro, E., Bardin, R., and Moiroud,
A. 1984. An improved method for lysis of Frankia with achromopeptidase allows detection of new plasmids. Can. J. Microbiol. 30: 12921295.
Simonet, P., Thi Le, N., Moiroud, A., and Bardin, R. 1989. Diversity of Frankia strains isolated from a single alder stand. Plant
Soil, 118: 1322.
Simonet, P., Grosjean, M.P., Misra, A.K., Nazaret, S., Cournoyer,
B., and Normand, P. 1991. Frankia genus-specific characterization by polymerase chain reaction. Appl. Environ. Microbiol.
57: 32783286.
Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221271.

1999 NRC Canada

J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:13 AM