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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
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)
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Strain*
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
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1264
Table 1. (concluded).
No.
57
Strain*
ACN14a
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
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.
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
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1265
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.
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
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1266
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
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.
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.
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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).
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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.
J:\cjb\cjb77\cjb-09\B99-083.vp
Monday, December 20, 1999 9:24:13 AM
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.
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