Nodulation speed of Frankia sp. on Alnus glutinosa, Alnus crispa, and Myrica gale
XAVIER NESME,' PHILIPPE NORMAND, FRANCINE M. TREMBLAY, AND MAURICE LALONDE~ De'partement des sciences forestiires, Universite' Laval, Que'bec (Que'.), Canada GIK 7P4
Received October 22, 1984
NESME, X., P. NORMAND, F. M. TREMBLAY, and M. LALONDE. 1985. Nodulation speed of Frankia sp. on Alnus glutinosa, Alnus crispa, and Myrica gale. Can. J. Bot. 63: 1292- 1295.
The question of compatibility between actinorhizal host plants and Frankia sp. was addressed using nodulation speed on Alnus spp. seedlings and Myrica gale seedlings. It was found that the speed of nodulation, defined as the mean time taken for the formation of the first prenodule, was a stable phenotype of both the Frankia strains and the host plants and that a distinction between slow-, medium-, and fast-nodulating Frankia strains could be made. The speed of nodulation of a given Frankia strain did not appear to be positively correlated to the original host plant from which isolation was first performed. It was, however, positively correlated with the Frankia strain and with the host plant species used for inoculation. Some optimal host plant - endophyte combinations were thus defined. Pure spore inocula of Frankia and in vitro propagated Alnus glutinosa plantlets were used to confirm that both the host plant and the microbial partners genetically influenced the nodulation process.
NESME, X., P. NORMAND, F. M. TREMBLAY et M. LALONDE. 1985. Nodulation speed of Frankia sp. on Alnus glutinosa, Alnus crispa, and Myrica gale. Can. J. Bot. 63: 1292- 1295.
Les auteurs ont CtudiC la compatibilitC entre des plantes h8tes actinorhiziennes et le Frankia sp. en examinant la vitesse de nodulation sur des plantules d'Alnus spp. et de Myrica gale. 11 est apparu que la vitesse de nodulation, dCfinie comme le temps moyen nCcessaire ti la formation du premier prC-nodule, est un phenotype stable a la fois des souches de Frankia et des plantes h6tes; il devient ainsi possible de distinguerdes souches de Frankia a nodulation lente, moyenne ou rapide. 11 ne semble pas y avoir de corrClation positive entre la vitesse de nodulation d'une souche donnCe de Frankia avec la plante h8te originale dont elle a CtC isolCe au depart. I1 y a cependant une corrClation positive entre la souche de Frankia et I'espbce de plante h6te sur laquelle elle est inoculCe. Ceci a permis de dCfinir quelques combinaisons endophyte - plante h8te optimales. Pour confirmer que le processus de nodulation est gCnCtiquement influencC a la fois par la plante h8te et par le microorganisme, des plantules d'Alnus glutinosa propagCes par culture de tissus ainsi que des inoculums de spores pures ont CtC utilisCs.
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Introduction The genetics of the actinorhizal symbiosis has been mainly
studied from the microsymbiont point of view or the host plant point of view, but rarely through the interaction of both part- ners. Moreover, the selection of bacterial strains or of host plant genotypes (Maynard 1980; Dawson and Sun 1981; Monaco et al. 1982; Normand and Lalonde 1982; Dillon and Baker 1982) has only focussed on the efficiency of nitrogen fixation. Nitrogen fixation, however, is not the only parameter whereby the symbiosis should be evaluated as demonstrated with the Rhizobium-legume symbiosis (Caldwell and Vest 1977; Williams 1981). Indeed, highly efficient strains must also be able to compete with wild strains to nodulate the host plant, resulting in a highly efficient association in the field.
The speed at which nodules are formed in a given combina- tion of bacterial strain and host plant would a means of evaluating the competitiveness of a strain with a given host plant and therefore their symbiotic compatibility. In the Rhizobium-legume symbiosis, such a parameter has been found to be genetically controlled by the host plant (Nutman 1949; Caldwell and Vest 1977) as well as by the microbial partner (DCnariC et al. 1976; Williams 198 1).
In actinorhizae, Lalonde and Quispel (1977) reported differ- ent nodulation speeds in cross inoculation between two Alnus species. This suggests the potential usefulness of the nod- ulation speed to evaluate the compatibility of an association. However, in cross-inoculation trials, the use of crushed nod- ules resulted in an inhibition of nodulation not noted when pure cultures were used (Lalonde 1979a; Vanden Bosch and Torrey 1983).
We therefore investigated the nodulation speed with pure
'Present address: Pathologie forestibre, lnstitut national de la re- cherche agronomique, route de Beaucouze, 49000 Angers, France.
'Author to whom all correspondence should be sent.
cultures of Frankia and three species of host plants. In addi- tion, the nodulation speed was also verified with spore sus- pensions of Frankia and with in vitro propagated clones of Alnus glutinosa.
Materials and methods Preparation of pure Frankia strains and Alnus glutinosa seedlings
The 22 Frankia strains (Table I) were grown on Qmod B liquid medium (Lalonde and Calvert 1979).
Seeds of A. glutinosa (L.) Gaertn. (Rhinelander, U.S.A.; gift of R. Hall) were surface sterilized and germinated in growth pouches (Lalonde 1979b). Conditions for growth and inoculation were as de- scribed previously (Normand and Lalonde 1982) except that the Frankia inoculant was treated with a drop of glacial acetic acid to eliminate CaC03 crystals and standardized to 5 pL packed-cell volume per seedling.
The 22 Frankia strains were tested on A. glutinosa with three replicates and ten 3-week-old seedlings per replicate.
Observations of the root systems were made daily from the 4th to the 1 1 t h day and every 2nd day thereafter, under the dissecting micro- scope. For each of the 30 seedlings, the time required for the appear- ance of the first prenodule as defined by Angulo Carmona (1974) was recorded.
Different combinations of three host-plants and of four Frankia strains All combinations of the three host plant species (Myrica gale L., A.
glutinosa, and Alnus crispa (Ait.) Pursh.) and of four Frankia strains (ACNIAG, AGNlg, Cpll , and MGPlOi) were assessed. The seeds of A. glutinosa and of A. crispa (Manic 5, P.Q.) were treated as de- scribed above and the seeds of M. gale (James Bay, P.Q.) treated with 500 ppm of gibberellic acid for 3 h (Torrey and Callaham 1979). The 12 combinations were tested with two replicates and twenty 3-week-old seedlings per replicate. The time of appearance of the first prenodule was recorded as above.
Pure spore inocula Four-week-old colonies of Frankia strains ACNI"", ARgN22d,
CpII, and MGPlOi were treated with 100 pL of 0.1 M citric acid to
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NESME ET AL
TABLE 1. Mean nodulation time of the Frankia strains tested on Alnus glutinosa seedlings
Original Mean (?SEM) time Strain host Reference" (days/seedlings)
ACN 10c A. crispa 1 7.098k0.338 ASP4f A. serrulata 2 7.124?0.049 CpI l C . peregrina 3 7.187&0.325 ARgN 14g A. rugosa 1 7.266k0.440 ACN5i A. crispa 1 7.315?0.447 ACN8a A. crispa 1 7.387k0.248 ACN 1 AG A. crispa 4 7.47020.163 ACN13h A. crispa 1 7.894L0.203 ACN 14a A. crispa 1 7.94320.472 ARgN9d A. rugosa 1 8.08820.206 TN 1 8aG Soil 1 8.131&0.164 ARgN8b A. rugosa 1 8.21820.277 ARgN22d A. rugosa 1 8.375k0.314 AGN l g A. glutinosa 5 8.430&0.242 ATPld A. tenuifolia 5 8.473kO. 154 AVP3n A. viridis 2 8.649?0.318 ACP 17b A. crispa 1 8.87420.205 ARbN4b A. rubra 5 8.99520.236 A R ~ P ~ A. rugosa 1 9.58320.240 ACN6j A. crispa 1 9.984k0.376 ARgN25c A. rugosa 1 10.15320.743 MGP l Oi M . gale 2 10.217k0.268
"References: I , Normand and Lalonde 1982; 2. Normand 1983; 3. Callaham er a/. 1978; 4, Lalonde 1979~; 5, Lalonde er a/. 1981.
eliminate CaC03 crystals, washed in phosphate-buffered saline, soni- cated and centrifuged in 95% (v/v) Percoll (Pharmacia, Sweden) at 37 000 X g for 20 min in a Beckman Airfuge. The spore suspension was harvested, adjusted to a density of lO\pores/mL of Crone's solution, and 1 mL was inoculated onto A. glutinosa seedlings grown in growth pouches. A pure culture inoculum prepared as usual was used as the control. Each treatment was replicated once for a total of about 60 seedlings per combination assessed.
Alnus glutinosa clones The in vitro propagated A. glutinosa clones AG-1 and AG-2
(PCrinet and Lalonde 1982) were grown according to Tremblay and Lalonde (1984). The in vitro rooted plantlets were washed in tap water, transferred to growth pouches, and inoculated with broken pure culture colonies of Frankia strains ARgN22d, CpII, and MGPlOi prepared as usual. Nonclonal seedlings from germinated seeds of A. glutinosa were used as controls of the infectivity of the inocula. Each Frankia strain was tested on 17 plantlets of AG-I, 14 plantlets of AG-2, and 30 control seedlings.
Controls In all experiments, control growth pouches containing seedlings
watered with sterile Crone's solution were kept among the inoculated ones.
Results Frankia strains tested on A. glutinosa
All 22 Frankia isolates listed in Table 1 nodulated 100% of the 71 8 A. glutinosa seedlings tested. Daily observations showed that the first prenodules appeared between 5 and 17 days after inoculation, with a normally distributed average of 8-10 days. There was a continuous gradient of nodulation speed among the strains from the fastest (ACNl Oc, 7.1 days) to the slowest (MGPlOi, 10.2 days). The 22 strains were