Plant and Soil 132: 207-211, 1991. 1991 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 8777
Diversity of Frankia strains isolated from single nodules of Alnus glutinosa
M. FAURE-RAYNAUD, C. DANIERE, A. MOIROUD and A. CAPELLANO Universit( Claude Bernard, Lyon I, UA 697, Ecologie Microbienne, 43 boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Received 22 June 1990. Revised October 1990
Key words: Alnus glutinosa, diversity, Frankia, isozyme
Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.
Actinorhizal plants fix dinitrogen through a sym- biotic relationship with an actinomycete of the genus Frankia. Since the first successful isolation of an infective and effective strain (Callaham et al., 1978) hundreds of Frankia strains have been isolated from numerous host plants including Alnus (Baker and Torrey, 1979; Normand and Lalonde, 1982; Faure-Raynaud and Moiroud, 1983), Casuarina (Diem et al., 1982), Elaeagnus (Baker and Torrey, 1980; Lalonde et al., 1981) Colletia (Gauthier et al., 1984) and Myrica (Saint Laurent et Lalonde, 1987). Many methods, detailed by Diem and Dommergues (1988), have been used for identification and classification of this large array of strains. Gel electrophoresis of soluble proteins have found to be useful in the identification of Frankia strains (Benson et al., 1984; Benson and Hanna, 1983; Gardes and Lalonde, 1987). Diversity in enzyme system activities has been also shown of interest for strain characterization (Faure-Raynaud et al., 1990; Gardes et al., 1987; Lalonde et al., 1988; Puppo et al., 1985; Steele and Stowers, 1986). All these investigations have demonstated large
Frankia strain diversity. Diversity has been ob- served among strains isolated from root systems of various actinorhizal species (Benson and Hanna, 1983; Simonet et al., 1989) and also among those isolated from a single alder plant (Faure-Raynaud et al., 1990; Simonet et al., 1985).
In a recent study performed to test if actino- rhizal host plants exercised selection pressure on the free populations of Frankia in soils we ob- tained about ninety isolates in pure culture. Among these isolates some originated from the same nodule.
In this paper are reported results concerning analysis of diversity among strains isolated from the same nodule using multienzyme method.
Material and methods
Isolation and cultivation
Frankia strains were isolated from nodules har- vested on the root systems of four Alnus glutin- osa Gaertn individuals differing in their flavonoid aglycones patterns (Gonnet and
208 Faure-Raynaud et al.
Dani~re, 1989) and growing in pots filled with a sandy soil collected in an alder stand. The Alnus glutinosa individuals were referred to 0, A, 13 and L. Isolation was performed from freshly excised nodules by using the OsO4 method (Lalonde et al., 1981). All nodule lobes were carefully separated and individually crushed in F medium (Faure-Raynaud et al., 1984). In every case all nodules and lobes were numbered. The following isolate designations were used: host species name (Ag), alder individual (0, 13, A, L), nodule number (1, 7, 12. . . ) and lobe num- ber (1 to 5). All strains were grown on F liquid medium added with Tween 80 in flasks and incubated for 3 to 4 weeks at 28C. Each flask containing 400 ml of medium was inoculated with 10 mL of Frankia suspension prepared from 18
Table I. Designation of Frankia strains isolated from 10 Alnus glutinosa nodules
Nodules Laboratory International designation designation a
1 Ag 13 11 ULF0107058011 Ag 13 12 ULF0107058012
2 Ag 13 71 U LF0107058071 Ag 13 72 ULF0107058072
3 Ag 0121 U LF0107058151 Ag 0122 ULF0107058152 Ag 0123 ULF0107058153
4 AgA 61 ULF0107058191 AgA 62 ULF0107058192 AgA 63 ULF0107058193 AgA 64 ULF0107058194 AgA 65 ULF0107058195
5 AgA 121 ULF0107058211 AgA 122 ULF0107058212
6 AgA 141 ULF0107058221 Ag A 142 U LF010705 8222 AgA 143 U LF0107058223 AgA 144 ULF0107058224 AgA 145 U LF010705 8225
7 AgA 161 U LF010705 8241 AgA 162 ULF010705 8242
8 AgL 141 ULF010705 8351 AgL 142 U LF010705 8352 AgL 143 U LF010705835 3 AgL 144 U LF010705 8354 AgL 145 U LF010705 8355
9 AgL 151 U LF0107058361 AgL 152 ULF0107058362
10 AgL 211 U LF0107058411 AgL 212 U LF0107058412 AgL 213 U LF0107058413 AgL 214 U LF0107058414
a Lechevalier, 1983.
to 20 days old colonies broken with a plastic syringe.
Thirty-two isolates originating from 10 differ- ent nodules were obtained (Table 1). Between two and five isolates were obtained from the same nodule.
Electrophoresis and enzyme system determination
Extracts were prepared for each strain as de- scribed previously (Faure-Raynaud et al., 1990) and tested for 4 enzyme systems: esterases (EST; EC 22.214.171.124), leucine aminopeptidase (LAP; EC 126.96.36.199), phosphoglucomutase (PGM; EC 188.8.131.52) and phosphoglucoisomerase (PG1; EC 184.108.40.206). Electrophoresis was performed on horizontal slab acrylamide agarose (Goullet, 1975) with Uriel migration buffer (1966). For each ge 1 25/xg of proteins, estimated by Brad- ford procedure (1976), were loaded in every well with bromophenol blue as marker dye. Elec- trophoretic separation of isozymes were carried out at 18C under a constant voltage of 150v until the marker dye has migrated for about 12 cm. Enzyme activities were detected in strain- ing solutions as described by Pasteur et al. (1987). To ascertain validity of the results, each enzyme determination was replicated at least three times.
The term electromorph designates the different patterns observed for each enzyme system. Esti- mates of electromorph diversity (H) were calcu- lated according to Gardes et al. (1987).
Results and discussion
Every strain showed activity for all enzyme sys- tems studied. Esterase activities were detected with a-naphthyl-acetate as substrate and esterase patterns showed high variation among strains: 5 different electromorphs were observed (Table 2). When LAP activity was analyzed all strains had a single fast band (Rf = 0.56) followed for some of them by two cathodal bands (Rf = 0.22 and 0.18) (Fig. 1). Two electromorphs were
noted (Table 2). Only one electromorph was detectable for PGI activity, characterized for all strains by a single band. Each strain had a single band pattern of PGM showing the same mobil ity (Fig. 1). One electromorph was noted (Table 2).
Thus six electromorphic types could be evi- denced from patterns corresponding to the 4 enzyme system activities exhibited by the Fran- kia isolates tested. So, out of the 32 isolates assayed six different strains were characterized.
All Frankia isolates originating from lobes of the same actinorhizae belonged to the same elec- tromorphic type (Table 2). So, they could be considered as reisolates of the same strain. This homology was observed even for esterase, the
Frankia strain diversity in single nodules 209
most discriminating enzyme system tested. On the other hand 6 actinorhizae out of the 10
harvested were induced by divergent strains (Table 2). It could be also noticed that actino- rhizae induced by the same Frankia strains were present on the root system of genetically differ- ent alder individuals (Table 2).
Electrophoretic separation of isozymes was de- monstrated as a resoluting method to differen- tiate closely related strains (Chun et al., 1985; Young, 1985). Isozymes studied have proved their validity for Frankia strain characterization (Faure-Raynaud et al., 1990; Gardes et al., 1987) and have been used to separate Frankia strains belonging to the same electrophoretic subgroups
Table 2. Electromorphs detected for the strains assayed and electromorph diversity calculated for the four enzyme systems tested
Nodules Strains Electromorphs a
EST LAP PGM PGI
1 Ag 13 1 1 5 1 3 3 Ag 13 1 2 5 1 3 3
2 Ag 13 7 I 2 2 3 3 Ag 13 7 2 2 2 3 3
3 Ag 0 12 1 4 I 3 3 Ag 0 12 2 4 1 3 3 Ag 0 12 3 4 1 3 3
4 AgA 61 3 2 3 3 AgA 62 3 2 3 3 AgA 63 3 2 3 3 AgA 64 3 2 3 3 AgA 65 3 2 3 3
5 AgA 121 4 1 3 3 Ag A 12 2 4 l 3 3
6 Ag A 14 1 3 2 3 3 Ag A 14 2 3 2 3 3 Ag A 14 3 3 2 3 3 Ag A 14 4 3 2 3 3 Ag A 14 5 3 2 3 3
7 AgA 161 3 2 3 3 Ag A 16 2 3 2 3 3
8 Ag L 14 1 1 2 3 3 Ag L 14 2 1 2 3 3 Ag L 14 3 1 2 3 3 Ag L 14 4 1 2 3 3 AgL 145 1 2 3 3
9 Ag L 15 1 2 2 3 3 AgL 152 2 2 3 3
10 Ag L 21 1 1 1 3 3 Ag L 21 2 1 1 3 3 Ag L 21 3 1 1 3 3 AgL 214 1 1 3 3
Electromorph number 5 2 1 1 Electromorph diversity (H) 0.736 0.431 0 0
a Electromorphs were numbered in order of decreasing anodal mobility (Gardes et al.. 1987).
210 Faure-Raynaud et al.
E lec t romorphs (N*) 1 2 3 4 5 1 2 ! 1
Enzymat ic sys tems EST LAP PGM PGI
Fig. I. Schematic representation of electromorphs deducted from EST (a-naph.), LAP, PGM and PGI zymograms pro- duced by electrophoresis of cell-fee extracts from 32 Frankia strains. Migration was toward the anode at the top.
(Gardes et al., 1987). In each of the 10 nodules tested we have not detected more than one strain. Moreover in some instances 5 isolates originating from the same nodule were analyzed.
In some previous studies it was claimed that actinorhizae could be induced by different Fran- kia strains. In Casuarinaceae, this possibility was deduced from the specificity and the effective- ness of the inocula used in some experiments (Redell and Bowen, 1985). However no strain was isolated from the nodules formed on these Casuarina root systems. The observations of Gauthier et al. (1981) on Casuarina and Hip- popha6 did not produce evidence in support of dual infection in Casuarina actinorhizae. On the other hand Benson and Hanna (1983) have dem- onstrated occupancy of one Alnus incana ssp rugosa nodule by two Frankia strains distinct by their morphology and their protein patterns. Dis- tinct strains in the same nodule have also been observed in Myrica pensylvanica by Bloom et al. (1988) and in Elaeagnus angustifolia by Dobritsa and Stupar (1989) using biomolecular tech- niques. Our results showed that the Alnus glutin- osa nodules tested were induced by a single strain. These observations do not rule out the possibilities of multiple strains presence in a nodule; they only give evidence of the scarceness of this eventuality.
The authors would like to thank Ms laumaud for technical assistance.
Baker D and Torrey J G 1979 The isolation and cultivation of actinomycetous root nodule endophytes. In Symbiotic Ni- trogen Fixation in the Management of Temperate Forest. Eds. J C Gordon, C T Wheeler and D A Perry. pp 38-56. Kluwer Academic Publishers, Dordreeht, The Nether- lands.
Baker D and Torrey J G 1980 Characterization of an effec- tive actinorhizal microsymbiont, Frankia sp Avcll (Ac- tinomycetales). Can. J. Microbioi. 26, 1066-1071.
Benson D R and Hanna D 1983 Frankia diversity in an alder stand as estimated by sodium dodecyl sulfate poly- arylamide gel electrophoresis of whole cell proteins. Can. J. Bot. 61, 2919-2923.
Benson D R, Buchholsz S E and Hanna D G 1984 Identifica- tion of Frankia strains by two dimensional polyaerylamide gel electrophoresis. Appl. Environ. Microbiol. 47, 489- 494.
Bloom R A, Mullin B C and Tate R L 1988 Strain variation among Frankia isolates from root nodules of Myrica pen- sylvanica (Bayberry). In 7th international meeting on Frankia and actinorhizal plants. Connecticut (communi- cation).
Bradford M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248-254.
Callaham D, Del Tredici P and Torrey J G 1978 Isolation and cultivation in vitro of the actinomycete causing root nodulation in Comptonia peregrina. Science 199, 899-902.
Chun P K, Sensabaugh G F and Vedros N A 1985 Genetic relationships among Neisseria species assessed by compara- tive enzyme electrophoresis. J. Gen. Microbiol. 131, 3105- 3115.
Diem H G and Dommergues Y R 1988 Isolation, characteri- zation and cultivation of Frankia. In Biological Nitrogen Fixation: Recent developments. Ed. N S Subba Rao. pp 227-254. Oxford and IBH publishing Co. Pvt. Ltd.
Diem H G, Gauthier D and Dommergues Y R 1982 Isolation and cultivation in vitro of an infective and effective strain of Frankia isolated from nodules of Casuarina sp. C.R. Ac. Sc. Paris 295 III, 759-762.
Dobritsa S V and Stupar O S 1989 Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizai plants. FEMS Microbiol. Letters 58, 287-292.
Faure-Raynaud M and Moiroud A 1983 Symbiose Frankia- Aulne: Culture in vitro de Frankia sp. sur diff6rentes sources de carbone et d'azote. C.R. Ac. Sc. Paris 296 III, 757-760.
Faure-Raynaud M, Bonnefoy M A, Perradin Y, Simonet P
and Moiroud A 1984 Protoplast formation from Frankia strains. Microbios 41, 159-166.
Faure-Raynaud M, Poirier-Bonnefoy M A and Moiroud A 1990 Diversity of Frankia strains isolated from actinorhizae of a single Alnus rubra cultivated in nursery. Symbiosis 8, 147-160.
Gardes M and Lalonde M 1987 Identification and subgroup- ing of Frankia strains using sodium dodecyl sulfate poly- acrylamide gel electrophoresis. Physiol. Plant. 70, 237- 244.
Gardes M, Bousquet J and Lalonde M 1987 Isozyme vari- ation among 40 Frankia strains. Appl. Environ. Microbiol. 53, 1596-1603.
Gauthier D L, Diem H G and Dommergues Y R 1981 Infectivit6 et effectivit6 de souches de Frankia isol6es de nodules de Casuarina equisetifolia et d'Hippopha~ rham- noides. C.R. Ac. Sc. Paris 293 III, 489-491.
Gauthier D L, Frioni L, Diem H G and Dommergues Y R 1984 The CoUetia spinosissima Frankia symbiosis. Oecol. Plant. 5, 231-239.
Gonnet J F and Dani~re C 1989 Individual variation in the flavonoid aglycones excreted on the leaves of Alnus glutin- osa (Betulaceae). Biochem. System. Ecol. 17, 239-247.
Goullet P H 1975 Esterase zymograms of Proteus and Pro- videncia. J. Gen. Microbiol. 87, 97-106.
Lalonde M, Calvert H E and Pine S 1981 Isolation and use of Frankia strains in actinorhizae formation. In Current Per- spectives in Nitrogen Fixation. Eds. A H Gibson and W E Newton. pp 296-299. Australian Academic Sciences, Can- berra.
Lalonde M, Simon L, Bousquet J and Seguin A 1988 Ad- vances in the taxonomy of Frankia: Recognition of species alni and elaeagni and novel subspecies pommerii and vandijkii. In Nitrogen Fixation: Hundred Years After. Eds. H Bothe, F J de Bruijn and W E Gustav. pp 671-680. Proceedings of the 7th International Congress on N =
Frankia strain diversity in single nodules 211
Nitrogen Fixation K61n (Cologne). Fischer, Stuttgart, New York.
Lechevalier M P 1983 Cataloging Frankia strains. Can. J. Bot. 61, 2964-2967.
Normand P and Lalonde M 1982 Evaluati...