Nodulation speed of Frankia sp. on Alnus glutinosa , Alnus crispa , and Myrica gale

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  • 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.

    [Traduit par le journal]

    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 arbi- trarily grouped into homogenous slow-, medium-, and fast- nodulating classes (Table 2). That classification of Frankia strains was found to be constant in the three replicates of the experiment.

    TABLE 2. Classification of the Frankia strains as a function of their nodulation

    speed on Alnus glutinosa

    Fast- Medium- Slow- nodulation nodulation nodulation

    class class class

    ACN10c ACN13h AVP3n ASP4f ACN14a ACP17b CpI l a ARgN9d ARbN4b ARgN 14g TN 1 8aG ARgP5x ACN5 i ARgN8b ACN6j ACN8a ARgN22d ARgN25c ACNIAG AGNIg MGPlOi

    ATPld

    TABLE 3. Mean nodulation time of four characteristic Frankia strains inoculated as pure cultures on three host plant species

    Frankia strains Host plant

    species CpI1 AGNlg ACNIA" MGP l Oi

    Myrica gale 7.536~ 8.27d 8.13cd 8.00bcd Alnus glutinosa 6.8 la 8.53d 7.576~ 9.99f Alnus crispa 8.52d 7.486 9.21e 11.00g

    NOTE: Values (days per seedling) are the means of two replicates with 9- 15 seedlings each. Means followed by different letters are significantly different at the 0.05 level with the LSD test.

    Combinations of host plants and Frankia strains Whatever the species of host plant tested (A. glutinosa, A.

    crispa or M. gale), all 295 seedlings produced their first pre- nodule between 5 and 16 days after inoculation with Frankia strains ACNIAG, AGNlg, Cpl l , and MGPlOi. A variance anal- ysis of the nodulation time indicated significant effects (0.01 level) for strains, host plants, and their interaction. As seen in Table 3, the combination of Cpll on A. glutinosa was signifi- cantly faster than all others, while that of MGP10i on A. crispa was the slowest.

    Pure spore inocula Pure spore inocula, compared with standard whole-colony

    inocula of strains ACNIAG, Cpl l , ARgN22d, and MGPlOi, had a slower nodulation speed (Table 4). Tbe relative nod- ulation patterns, however, were maintained; in both cases Cpll was the fastest and MGPlOi was the slowest. MGPlOi again was markedly different from the other three strains, the per- centage of nodulated seedlings being only 75% after 26 days instead of the constant 100% with the three other strains.

    Clones of A. glutinosa The utilization of A. glutinosa clones compared with seed-

    lings did not affect the nodulation speed of strains Cpll , ARgN22d, and MGP lOi (Table 5). Variance analysis showed that the 1 day difference between clones in the nodulation time was significant at the 0.01 level.

    Discussion All the 22 Frankia isolates tested formed prenodules and,

    later on, formed true nodules on all inoculated seedlings. As described previously (Lalonde and Quispel 1977), no pre- nodule appeared before a 5-day period. The peak in prenodule appearance was 8- 10 days after inoculation and agrees with that previously reported (Normand and Lalonde 1982). The

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  • CAN. J. BOT. VOL. 63, 1985

    TABLE 4. Mean nodulation time of four characteristic Frankia strains inoculated a broken- colony inocula or as pure spore inocula on A. glutinosa seedlings

    Frankia strain Type of inoculum CpI 1 ACN 1 ARgN22d MGP 1 Oi Inocula average

    Broken colonies 7.44k0.11 8.06k0.16 8.79k0.16 10.56k0.19 8.7 1 Pure spores 8.27k0.11 8.49kO. 1 1 9.27kO. 11 > 16" > 10.5

    Strain average 7.85 8.27 9.03 >13

    NOTE: Values (days per seedling) are means ( S E M ) of 57-66 seedlings in two replicates. "Only 75% of the plants were nodulated after 26 days; therefore, this mean value is only an approximation calculated on

    the 75% nodulated seedlings.

    TABLE 5. Mean (+SEM) nodulation time (days per plant) of three characteristic Frankia strains inoculated on A. glutinosa seedlings or

    in vitro propagated clones after transfer in growth pouches

    Frankia strain Type of Clone

    host plant CpI 1 ARgN22d MGP 10i average

    Seedlings 7.46k0.19 8.73k0.18 10.97k0.27 9.05 Clone AG-1 7.00k0.21 7.64k0.36 10.64k0.49 8.43 Clone AG-2 8.41 k0.23 8.94k0.23 11.3820.33 9.57

    Strain average 7.62 8.44 10.99

    present report constitutes the first detailed study of the nod- ulation speed parameter, following Maynard's (1980) obser- vation that crushed-nodule provenances delayed nodulation rel- ative to pure cultures. The relative nodulation speed of the Frankia strains was consistent throughout the experiment, indi- cating that the nodulation speed phenotype is genetically con- trolled by the microsymbiont. This extends to the Frankia symbiosis observations reported for Rhizobium, which nor- mally forms nodules 5- 10 days after inoculation (DCnariC and Truchet 1976; Williams 198 1).

    The Alnus species from which a given Frankia strain was originally isolated does not affect the nodulation speed of that strain on A. glutinosa, since Frankia strains isolated from both A. crispa and A. rugosa occurred in all nodulation speed classes. Furthermore, when the Frankia strains were iso- lated from various Alnus species such as A. crispa, A. rugosa, A. serrulata, or A. tenuifolia, their nodulation speed was not delayed relative to the standard combination of AGNlg on A. glutinosa. These results differ from those of Lalonde and Quispel (1977), who found an effect from nodule donor host plant species, but they were working with crushed nodules rather than with pure cultures.

    Cross-inoculation tests between four Frankia strains and three host plant species indicate that nodulation speed is specific to any given Frankia strain - host plant combination. The strongest interaction was observed with MGPlOi, which was different from the three other strains tested. MGPlOi had a normal nodulation speed on M. gale, but a very slow nod- ulation speed on the two Alnus species. This represents a form of partial incompatibility that would be acting during the first events of the interaction between the plant and the bacterium. This partial incompatibility detected with the MGPlOi strain isolated from M. gale is consistent with results obtained with crushed-nodule inocula. Indeed, crushed M. gale nodule inocula have been found to result in ineffective nodules or simply failed to induce nodulation in other Myrica species (Mackintosh and Bond 1970; Miguel et al. 1978; Vanden

    Bosch and Torrey 1983) or in Alnus species (Rodriguez- Barmeco and Bond 1976). Differences in compatibility be- tween Frankia and host plant have also been described, but at the level of nitrogenase activity (Dillon and Baker 1982).

    To confirm the results obtained with a standard inocula of broken colonies that comprise vesicles and hyphae as well as spores, we used suspensions of spores of a constant density. The results obtained with broken colonies were confirmed with purified spores, indicating that the nodulation speed is genet- ically controlled by the microsymbiont. A germination lag phase of the spores does probably explain the slight delay observed relative to the nodulation speed with broken colonies.

    Significant differences were found between two A. glutinosa clones, confirming the existence of a genetic control of the nodulation speed by the host plant. This intraspecific control was recognized by Maynard (1980), who used two prov- enances of A. glutinosa seeds. This underlines the value of selecting fast-nodulating, actinorhizal host plant clones. Such a selection has already been done for Trifolium sp. (Hely 1972), resulting in an increased biomass-accretion potential.

    The partial strain - host plant compatibility illustrated by different nodulation speeds points out the equal importance of both partners in a successful symbiotic association. The nod- ulation speed assay, standardized pure spore inocula, and in vitro propagated clones may prove valuable in the genetic study of the interaction between Frankia and actinorhizal species. They may also be suitable tools to select fast-nodulating part- ners that if coupled with highly efficient symbioses, would result in a high biomass accretion.

    Acknowledgements X.N. and F.M.T. received financial supports from ENFOR

    (project Nos. P- 198 and P-232), P.N. received a Fonds For- mation des chercheurs et d'action concertke scholarship, and M.L. received a Natural Sciences and Engineering Research Council of Canada grant (No. U-0209).

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