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

Abstract

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.

Introduction

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, 1 2 . . . ) 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 28°C. 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 U L F 0 1 0 7 0 5 8 0 1 1 Ag 13 12 U L F 0 1 0 7 0 5 8 0 1 2

2 Ag 13 71 U L F 0 1 0 7 0 5 8 0 7 1 Ag 13 72 U L F 0 1 0 7 0 5 8 0 7 2

3 Ag 0121 U L F 0 1 0 7 0 5 8 1 5 1 Ag 0122 U L F 0 1 0 7 0 5 8 1 5 2 Ag 0123 U L F 0 1 0 7 0 5 8 1 5 3

4 AgA 61 U L F 0 1 0 7 0 5 8 1 9 1 AgA 62 U L F 0 1 0 7 0 5 8 1 9 2 AgA 63 U L F 0 1 0 7 0 5 8 1 9 3 AgA 64 U L F 0 1 0 7 0 5 8 1 9 4 AgA 65 U L F 0 1 0 7 0 5 8 1 9 5

5 AgA 121 U L F 0 1 0 7 0 5 8 2 1 1 AgA 122 U L F 0 1 0 7 0 5 8 2 1 2

6 AgA 141 U L F 0 1 0 7 0 5 8 2 2 1 Ag A 142 U L F 0 1 0 7 0 5 8 2 2 2 AgA 143 U L F 0 1 0 7 0 5 8 2 2 3 AgA 144 U L F 0 1 0 7 0 5 8 2 2 4 AgA 145 U L F 0 1 0 7 0 5 8 2 2 5

7 AgA 161 U L F 0 1 0 7 0 5 8 2 4 1 AgA 162 U L F 0 1 0 7 0 5 8 2 4 2

8 AgL 141 U L F 0 1 0 7 0 5 8 3 5 1 AgL 142 U L F 0 1 0 7 0 5 8 3 5 2 AgL 143 U L F 0 1 0 7 0 5 8 3 5 3 AgL 144 U L F 0 1 0 7 0 5 8 3 5 4 AgL 145 U L F 0 1 0 7 0 5 8 3 5 5

9 AgL 151 U L F 0 1 0 7 0 5 8 3 6 1 AgL 152 U L F 0 1 0 7 0 5 8 3 6 2

10 AgL 211 U L F 0 1 0 7 0 5 8 4 1 1 AgL 212 U L F 0 1 0 7 0 5 8 4 1 2 AgL 213 U L F 0 1 0 7 0 5 8 4 1 3 AgL 214 U L F 0 1 0 7 0 5 8 4 1 4

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 3.1.1.1), leucine aminopeptidase (LAP; EC 3.4.11.1), phosphoglucomutase (PGM; EC 2.7.5.1) and phosphoglucoisomerase (PG1; EC 5.3.1.9). 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 18°C 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.

Numerical analysis

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). On ly one e lec t romorph was

detec table for P G I activity, character ized for all

s trains by a single band . Each strain had a single band pa t t e rn of P G M showing the same mobi l i ty (Fig. 1). One e lec t romorph was no ted (Table 2).

Thus six e lec t romorphic types could be evi- denced f rom pat te rns cor responding to the 4

enzyme system activities exhibi ted by the Fran- kia isolates tested. So, out of the 32 isolates

assayed six different strains were characterized.

All Frankia isolates or ig inat ing from lobes of the same act inorhizae be longed to the same elec-

t romorphic type (Table 2). So, they could be cons idered as reisolates of the same strain. This homology was observed even for esterase, the

Frank ia strain diversity in single nodules 209

most d iscr iminat ing enzyme system tested. O n the o ther hand 6 act inorhizae out of the 10

harvested were induced by divergent strains

(Table 2). It could be also not iced that act ino- rhizae induced by the same Frankia strains were

present on the root system of genetical ly differ- ent alder individuals (Table 2).

E lec t rophore t ic separa t ion of isozymes was de-

mons t ra ted as a resolut ing me thod to differen-

tiate closely re la ted strains ( C hun et al., 1985;

Young, 1985). Isozymes studied have proved their validity for Frankia strain character iza t ion

( F a u r e - R a y n a u d et al., 1990; Gardes et al., 1987) and have been used to separate Frankia strains

be longing to the same e lect rophoret ic 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 A g A 61 3 2 3 3 A g A 62 3 2 3 3 A g A 63 3 2 3 3 A g A 6 4 3 2 3 3 A g A 65 3 2 3 3

5 A g A 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 A g A 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.

0.50

I

0.25

Anode

b

I I

E l e c t r o m o r p h s (N* ) 1 2 3 4 5 1 2 ! 1

E n z y m a t i c s y s t e m s EST L A P P G M P G I

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 i t 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.

Acknowledgement

The authors would like to thank Ms laumaud for technical assistance.

N Guil-

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