6
Axenic nodulation of in vitro propagated Alnus glutinosa plantlets by Frankia strains PIERRE P~RINET' AND MAURICE LALONDE' Dipartement d'Ecologie er Pe'dologie, Faculre' de Foresterie er Ge'orle'sie, Universite' Laval, Que'bec (Que'.), Canada GIK 7P4 Received December 1, 1982 P~RINET, P., and M. LALONDE. 1983. Axenic nodulation of in vitro propagated Alnus glurinosa plantlets by Frankia strains. Can. J. Bot. 61: 2883-2888. Two clones of Alnus glurinosa plantlets propagated in virro were inoculated, under axenic conditions, with eight different isolates of Frankia originating from different provenances and representing both type-N and type-P strains. All the plants inoculated formed typical and effective actinorhizae. The nodulated root systems were tested for the presence of microbial contaminants on different culture media. Typical Frankia colonies, growing on the root pieces incubated in the culture media, were subcultured and reinoculated on alder seedlings grown in plastic growth pouches. 'The results demonstrated that Frankia can induce a typical and effective nodulation on axenic Alnus glutinosa host plants without the help of any "microbial helper". P~RINET, P., et M. LALONDE. 1983. Axenic nodulation of in virro propagated Alnus glurinosa plantlets by Frankia strains. Can. J. Bot. 61: 2883-2888. On inocule en conditions axCniques des plantules de deux clones d'Alnus glurinosa, issues de multiplication vCgCtative in virro, avec huit isolats differents de Frankia. Ces souches de Frankia sont isolCes de sept plantes-h6tes provenant de 4 pays diffkrents et qui repkentent deux types de souche: le type N et le type P. Toutes les plantes inoculCes foment des actinorhizes typiques capables de fixer l'azote atmosphCrique. Pour verifier l'axCnie du systkme, on incube le systkme racinaire nodule sur diffkrents milieux de culture. Des colonies typiques de Frankia se dCveloppent sur les sections de racines inoculCes sur les milieux de culture. Des colonies de Frankia sont repiquCes et rCinoculCes sur des semis d'aulne glutineux en sachets de croissance. Les rCsultats dCmontrent que Frankia peut induire une nodulation typique et fonctionnelle sur des plants d'Alnus glutinosa en conditions axCniques, i.e., en absence de tout autre microorganisme. Introduction Numerous Frankia isolates obtained from different actinorhizal host plants are now available in pure cultures (Callaham et al. 1978; Quispel and Tak 1978; Baker, Kidd, and Torrey 1979; Baker, Torrey , and Kidd 1979; Beny and Torrey 1979; Quispel 1979; Burggraaf et al. 1981; Lechevalier 1981; Diem et al. 1982; Normand and Lalonde 1982) and could be used for the study of the nodulation process when inoculated to host plants in axenic conditions. Knowlton et al. (1980) reported the requirement of some "microbial helper" for the successful infection of the actinorhizal host plant Alnus rubra. Although Lalonde et al. (198 1) reported the axenic nodulation of Alnus glutinosa seedlings inoculated with Frankia strain AGNlg without the presence of any "microbial helper," this subject is still controversial. with seedlings produced from surface-sterilized seeds. We present the results of the successful nodulation of Alnus glutinosa plantlets propagated in vitro and inocu- lated by eight different strains of Frankia in axenic conditions, i.e., in the absence of any detectable "micro- bial helper. " Material and methods Alnus glutinosa plantlets Alnus glutinosa plantlets were obtained from in vitro propagation by tissue culture techniques (PCrinet and Lalonde 1983). Multiple shoots were formed from seedling cultures of A. glurinosa; the shoots were multiplied in the presence of benzyladenine (BAP) at a concentration of 1-5 pm. Individual shoots were rooted in vitro with indolebutyric acid (IBA) at 1 p m Rooted plantlets 10-15 mrn high of two clones of A. glutinosa (AG-1 and AG-2) were used for the nodulation tests, but most of them were from clone AG-1 (second experiment). TO demonstrate the ability of other Frankia strains to Fra&a at loo' aseptic host plants, we used The eight different strains of Frankia used, representing glutinosa plantlets propagated in vitro. The in vitro both type-P and type-N strains (Normand and Lalonde 1982), propagation of A. glutinosa permitted us to obtain a were isolated from different host plants (Table 1). All Frankia large number of plantlets already free of microorgan- strains were maintained in pure culture in the QmodB medium isms in a relatively short period of time in comparison of Lalonde and Calvert (1979) containing 5 mg/L of a-lecithin and at a pH of 6.8-7.0. For the strain A R ~ P ~ @ , 65 mL/L of 'Present address: Jardin Botanique de Montreal, 4101 fetal bovine serum (FBS, Gibco Co., NY, catalogue No. Sherbrooke E., MontrCal (QuC.), Canada HlX 2B2. 240-1555) was added to the QmodB medium (Lalonde and 'Supported by the Natural Sciences and Engineering Re- Calvert 1979). Colonies of Frankia contained in glass tubes search Council of Canada grant No. A7192. (20 X 150 mm) of 15 mL of QmodB medium were harvested at Can. J. Bot. Downloaded from www.nrcresearchpress.com by SAVANNAHRIVNATLABBF on 11/14/14 For personal use only.

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Page 1: Axenic nodulation of               in vitro               propagated               Alnus glutinosa               plantlets by               Frankia               strains

Axenic nodulation of in vitro propagated Alnus glutinosa plantlets b y Frankia strains

PIERRE P~RINET ' AND MAURICE LALONDE' Dipartement d'Ecologie er Pe'dologie, Faculre' de Foresterie er Ge'orle'sie, Universite' Laval, Que'bec (Que'.),

Canada G I K 7P4 Received December 1, 1982

P ~ R I N E T , P., and M. LALONDE. 1983. Axenic nodulation of in vitro propagated Alnus glurinosa plantlets by Frankia strains. Can. J. Bot. 61: 2883-2888.

Two clones of Alnus glurinosa plantlets propagated in virro were inoculated, under axenic conditions, with eight different isolates of Frankia originating from different provenances and representing both type-N and type-P strains. All the plants inoculated formed typical and effective actinorhizae. The nodulated root systems were tested for the presence of microbial contaminants on different culture media. Typical Frankia colonies, growing on the root pieces incubated in the culture media, were subcultured and reinoculated on alder seedlings grown in plastic growth pouches. 'The results demonstrated that Frankia can induce a typical and effective nodulation on axenic Alnus glutinosa host plants without the help of any "microbial helper".

P~RINET, P., et M. LALONDE. 1983. Axenic nodulation of in virro propagated Alnus glurinosa plantlets by Frankia strains. Can. J. Bot. 61: 2883-2888.

On inocule en conditions axCniques des plantules de deux clones d'Alnus glurinosa, issues de multiplication vCgCtative in virro, avec huit isolats differents de Frankia. Ces souches de Frankia sont isolCes de sept plantes-h6tes provenant de 4 pays diffkrents et qui repkentent deux types de souche: le type N et le type P. Toutes les plantes inoculCes foment des actinorhizes typiques capables de fixer l'azote atmosphCrique. Pour verifier l'axCnie du systkme, on incube le systkme racinaire nodule sur diffkrents milieux de culture. Des colonies typiques de Frankia se dCveloppent sur les sections de racines inoculCes sur les milieux de culture. Des colonies de Frankia sont repiquCes et rCinoculCes sur des semis d'aulne glutineux en sachets de croissance. Les rCsultats dCmontrent que Frankia peut induire une nodulation typique et fonctionnelle sur des plants d'Alnus glutinosa en conditions axCniques, i.e., en absence de tout autre microorganisme.

Introduction Numerous Frankia isolates obtained from different

actinorhizal host plants are now available in pure cultures (Callaham et al. 1978; Quispel and Tak 1978; Baker, Kidd, and Torrey 1979; Baker, Torrey , and Kidd 1979; Beny and Torrey 1979; Quispel 1979; Burggraaf et al. 1981; Lechevalier 1981; Diem et al. 1982; Normand and Lalonde 1982) and could be used for the study of the nodulation process when inoculated to host plants in axenic conditions.

Knowlton et al. (1980) reported the requirement of some "microbial helper" for the successful infection of the actinorhizal host plant Alnus rubra. Although Lalonde et al. (198 1) reported the axenic nodulation of Alnus glutinosa seedlings inoculated with Frankia strain AGNlg without the presence of any "microbial helper," this subject is still controversial.

with seedlings produced from surface-sterilized seeds. We present the results of the successful nodulation of Alnus glutinosa plantlets propagated in vitro and inocu- lated by eight different strains of Frankia in axenic conditions, i.e., in the absence of any detectable "micro- bial helper. "

Material and methods Alnus glutinosa plantlets

Alnus glutinosa plantlets were obtained from in vitro propagation by tissue culture techniques (PCrinet and Lalonde 1983). Multiple shoots were formed from seedling cultures of A . glurinosa; the shoots were multiplied in the presence of benzyladenine (BAP) at a concentration of 1-5 pm. Individual shoots were rooted in vitro with indolebutyric acid (IBA) at 1 p m Rooted plantlets 10-15 mrn high of two clones of A. glutinosa (AG-1 and AG-2) were used for the nodulation tests, but most of them were from clone AG-1 (second experiment).

TO demonstrate the ability of other Frankia strains to Fra&a at loo' aseptic host plants, we used The eight different strains of Frankia used, representing

glutinosa plantlets propagated in vitro. The in vitro both type-P and type-N strains (Normand and Lalonde 1982), propagation of A. glutinosa permitted us to obtain a were isolated from different host plants (Table 1). All Frankia large number of plantlets already free of microorgan- strains were maintained in pure culture in the QmodB medium isms in a relatively short period of time in comparison of Lalonde and Calvert (1979) containing 5 mg/L of a-lecithin

and at a pH of 6.8-7.0. For the strain A R ~ P ~ @ , 65 mL/L of 'Present address: Jardin Botanique de Montreal, 4101 fetal bovine serum (FBS, Gibco Co., NY, catalogue No.

Sherbrooke E., MontrCal (QuC.), Canada HlX 2B2. 240-1555) was added to the QmodB medium (Lalonde and 'Supported by the Natural Sciences and Engineering Re- Calvert 1979). Colonies of Frankia contained in glass tubes

search Council of Canada grant No. A7192. (20 X 150 mm) of 15 mL of QmodB medium were harvested at

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2884 CAN. J. BOT. VOL. 61. 1983

TABLE 1. Description of the Frankia strains used for the axenic nodulation of Alnus glutirzosa

Frankia Strain strain Host plant Origin source*

Type N ACN 1 Alnrcs crispa Quebec 1 ARbN4b Alr~us rubra Oregon 2 AGN 1 g Alnus glutirzosa Holland 2 ARgN 18f Alnrcs rrcgosa Quebec 3

Type P AVP3f Alrrus viridis France 4 A R ~ P ~ ~ Alnus rrcgosa Quebec 3 ATP 1 d Alnus tenuifolia Colorado 2 MgP 10i Myrica gale Quebec 4

*I , Lalonde and Calven 1979; 2. Lalonde er a / . 1981; 3, Normand and Lalonde 1982; 4, P. Normand and M. Lalonde, unpublished results.

TABLE 2. Age of Frankia inoculum used for the axenic nodulation of Alnus glutinosa

Age of inoculum, Experiment no, weeks series no.

TY pe N ACN i ARbN4b AGN 1 g ARgN 18f

Type P AVP3f AR~PsA--G ATP 1 d MgP 1 Oi

different ages (Table 2); two colonies were transferred through 10mL of sterile phosphate buffer saline (PBS) at pH 7.2. Frankia colonies were homogenized in the PBS through repeated passages into a No. 21 needle with a sterile syringe. A volume of 1 mL of the homogenized sus~ension of broken .. hyphae and spores was directly inoculated to each plantlet immediately after its transfer to a culture vessel. The Frankia inoculant was applied at the rate of one colony of Frankia for five plants of A. glutinosa.

Cultural conditions Nodulation tests were performed in two different series of

experiments. In the first series, Erlenmeyer flasks of 1 L were used for the nodulation and growth of the A. glutinosa plantlets. Flasks contained 325 mL of an artificial substrate (vermiculite-Turface, 1 : 1 , v /v) and 180 m L of N-free Crone's solution (Lalonde 1979). Flasks were closed with foam plugs and autoclaved at 121°C and 15psi (1 psi = 6.894 757 kPa) for 60 min. After transfer and inoculation of the plantlets into the flasks, they were incubated in a growth chamber at a day temperature of 24°C and 20°C at night, with a light intensity of 10 000 lx, a photoperiod of 16 h, and a relative humidity of 70% or more.

In the second series of experiments, three different sizes of glass culture tubes were used: 23 X 200 mm, 35 X 200 mm, and 35 X 300 mm. Culture tubes of each size contained, respectively, 30 mL of substrate (vermiculite-Turface, 1 : 1) and 15 mL of N-free Crone's solution, 80 mL of substrate and 40 mLof Crone's solution, and finally 120 mLof substrate and 60 mL of Crone's solution. Tubes were closed with foam plugs and autoclaved for 30-40 min at 121°C and 15 psi. All culture tubes were incubated on shelves in the laboratory under fluorescent lamps (Gro-lux and cool-white fluorescent lamps, Sylvania) at 5500 lx. The temperature was 27-32°C and the photoperiod was 16 h.

The pH of the vermiculite-Turface substrate with the N-free Crone's solution after the sterilization was 5.5 and stayed constant throughout the experiment.

Detection o f contaminants The vermiculite-~urface substrate was sampled after the

sterilization of the culture vessel and incubated in the QmodB medium. Shoots of A. glutinosa were excised from the in vitro proliferating mass on the multiplication medium, prior to the rooting treatment, and transferred into tubes of QmodB medium. All Frankia inoculants were also tested in the QmodB medium after the inoculation of the plantlets.

After 6 weeks of growth, all the plants were removed from their flasks, the root systems were sectioned, and root samples were transferred aseptically onto different culture media (Table 3) to detect the presence of eventual contaminants.

In the first series of experiments, the roots of each plant were incubated in 10 tubes of QmodB medium. After 2-3 weeks, aliquots of the QmodB medium, from the tubes incubated with the root samples and the nodules, were inoculated on different culture media in Petri dishes (Table 3). 'The plates were incubated in aerobic conditions and also in anaerobic jars. In the second series of experiments, the roots of each plant were incubated directly in both the liquid and the solid media. For each plant, root samples and root nodules were inoculated into two tubes of QmodB, one of SMB, and 1 Petri dish of each solid medium. Root samples were also inoculated on one plate of SMA medium incubated in anaerobic conditions.

Effectivity of the nodules The effectivity of the nodules was confirmed by the

acetylene-reduction assay. Roots with nodules were incubated

TABLE 3. Culture media used for the incubation of the nodules and samples of the roots for the detection of microbial con-

taminants

Liquid media QModB* t SMBt

Solid media SMA*? PDA* t MPH* t CZK*t MA* CM*

Lalonde and Calvert 1979 Stand methods broth, BBL

Standard methods agar, BBL Potato dextrose agar, Difco Milk protein hydrolysate agar, BBL Czapek solution agar, Difco Malt agar, Difco Cornmeal agar, Difco

*Used in experiment series I . tUsed in experiment series 2.

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PERINET AND LALONDE

TABLE 4. Nodulation of A111us glutinosa plantlets and percent of axeny of the nodulated plants

% of % axenically

Frankia Experiment NO. nodulated nodulated strain series no." plants plants Effectivity? plants

TY pe N ACN 1 AG 1 8 100 + 50

1 6 100 + 83.4 1 4 100 + 100 2 12 100 + 33.3 2 5 100 NDS 80

ARbN4b 2 19 100 + 5 8 AGN 1 g 1 5 100 + 100

2 18 72 + 100 ARgN 18f 1 5 100 + 100

Type P AVP3f 2 15 100 + 86.6 AR~PSE 2 5 loo + 20 ATP 1 d 2 20 95 ND 84 MgP 1 Oi 2 20 95 ND 5

Total 142

* I , in Erlenmeyer flasks, clones AG-I and AG-2; 2, i n culture tubes, clone AG-I +As determined by the acetylene-reduction assay. SND, not determined.

in 10% (v/v) acetylene in air. Samples of the gaseous phase were analysed for C2HJ at different incubation times with a Hewlett-Packard gas chromatograph (model 57 10A) equipped with FID and a 2 mm x 1.8 m Porapak N, 80- to 100-mesh, filled column. The injection port, column, and detector temperatures were 100,50, and 120°C and the flow rates of Hz, air, and N2 carrier gas were 20, 200, and 30mL.min-', respectively.

Subculture of the Frankia and nodulation in growth poriches The colonies of Frankia A C N ~ ~ ~ that were growing from

the nodules and on the roots in the QmodB medium were subcultured twice in the same QmodB medium and inoculated on seedlings of Alnus glutinosa grown in plastic growth pouches in the N-free Crone's solution, according to Lalonde and Calvert (1979).

Results Nodulation

The nodulation test on Alnus glutinosa plantlets inoculated with Frankia gave, after 6 weeks of growth, a percentage of nodulation of 100% for six strains and of 95% for the two other Frankia strains (Table 4). Out of 15 1 plantlets initially inoculated, 9 plantlets died. From the 142 remaining plantlets, 135 formed nodules and only 7 did not nodulate. These 7 plantlets, though still alive after 6 weeks, had not grown normally compared with the uninoculated control plants. ~ s e ~ t i c plantlets from both clones AG-1 and AG-2 (Table 4) formed nodules when inoculated with pure cultures of Frankia.

The root nodules were visible after 2 weeks. In

Erlenmeyer flasks the nodulated plantlets grew larger than the control plants left uninoculated (Fig. 1). The control plantlets did not nodulate, but their leaves were still green after 6 weeks owing to previous nitrogen nutrition from the plant tissue culture medium. Typical lobed nodules were formed on the upper part of the root system (Fig. 2).

Light microscopy of the nodules indicated a typical development of the Frankia endophyte for all the strains tested in the cortical cells with normal formation of septate vesicles. The effectivity of the actinorhizae or root nodules was confirmed by the acetylene-reduction assay, which averaged for the six strains tested an activity of 20-64nmol C2H4.h-'.mg nodule dry weight- l .

Detection of eventual contaminants The incubation in the QmodB medium of all the initial

samples of the substrate, the shoots of A. glutinosa, and the Frankia inoculants confirmed that they were free of microbial contaminants.

After 6 weeks of growth, the root systems of all the plantlets were tested for the presence of microbial contaminants in different culture media listed inTable 3. When present, contaminants were mostly bacteria, few of them being fungi.

For each strain of Frankia tested, nodulation of plantlets of Alnus glutinosa was demonstrated to be feasible without the help of any microbial helper. The

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2886 CAN. J . BOT.

percentage of nodulated plants that was still axenic after 6 weeks was most often 80-100% (Table 4). When a contaminant was present on the nodules and (or) the root pieces, the bacteria and (or) the fungi grew in less than a week. In the absence of any contaminant, typical Frankia colonies, introduced as the Frankia inoculant, started to grow after 2 weeks on the nodules and (or) the root pieces incubated in the QmodB, SMB, and SMA media (Fig. 4). Hyphae, vesicles, and sporangia were easily observed in the colonies. The microscopic deter- mination using interference phase optics confirmed the purity of the reisolated Frankia cultures.

As a confirmation of the identity of the isolates, colonies of Frankia ACN 1 AG, which developed from nodules and root pieces incubated in the QmodB medium, were subcultured twice and their infectivity was tested on seedlings of Alnus glutinosa grown in plastic growth pouches. These nodulation tests of the reisolated cultures of Frankia ACNIAG gave a per- centage of nodulation of 100%.

Discussion The results demonstrate that the eight different strains

of Frankia tested can easily nodulate aseptic host plants. Actually there is no evidence that a particular Frankia strain could require the help of another microorganism to infect the Alnus glutinosa host plant. The eight different Frankia strains used were isolated from seven different species of host plant from Canada, U.S.A., France, and Holland and tested on the European species A. glutinosa. The Frankia strains used were of both the type N and the type P according to Normand and Lalonde (1982). In spite of that variability in provenance and in strain type, all of them were able to infect aseptic plants of this host. The nodulation was not affected by the age of the Frankia inoculants, which were obtained from 3- to 20-week-old colonies.

The optimum pH for the growth of nodulated A. glutinosa is around 5.4 (Bond et al. 1954) and Frankia development in vitro is optimum within a range pH of 6-8 (Burggraaf and Shipton 1982). The QmodB medium used had a pH of 6.8 and the artificial substrate utilized with the N-free Crone's solution for the nodula- tion tests had a pH of 5.5, and this corresponds to the optimum pH for the growth of nodulated alder plants.

Different factors act together to explain the lack of nodule formation for one plantlet out of 20 inoculated with strains ATPld and MgPlOi. The in vitro biomass development of type-P strain is less than for type N (Normand and Lalonde 1982), so the concentration of inoculant was lower for these strains. Finally, the cultural conditions prevailing in experiment series 2, compared with series 1, emphasize the influence of environmental conditions on the receptivity of the host

VOL. 61. 1983

plants. In the first series of experiments, in Erlenmeyer flasks incubated in a growth chamber, the adaptation of the plantlets to the n e b environment was facilitated by a better control of the relative humidity and the tempera- ture. Also growth of the plants was better, more space was available for both theroots and the leaves, and iight intensity was higher. In the second series of experi- ments, in culture tubes incubated on shelves under fluorescent lamps in the laboratory, the relative humid- ity dropped rapidly and the temperature frequently reached over 30°C. These two factors contributed to the rapid drying of the substrate and consequently to a decrease of the water potential, which in turn, at low values, has a deleterious effect on Frankia growth and could limit the infection of the root by Frankia as suggested by Shipton and Burggraaf (1982). In addition to these higher stress conditions, space and light intensity limited the growth of the plantlets in the tubes (Fig. 3). Therefore the nodulation was 100% for the first series of experiments and 94% for the second series. Plantlets inoculated with AGN lg that did not nodulate were probably less receptive owing to stress conditions of low humidity and high temperature prevailing in the second series of experiments.

Typical clusters of septate vesicles and hyphae were observed in the nodules for all the strains tested and the acetylene-reduction assay, performed for six Frankia strains, confirmed the effectivity of the actinorhizae.

The percentage of axeny of the nodulated plants averaged 80- 100% for most of the Frankia strains with a range of 5 to 100% (Table 4). In spite of the fact that all the individual elements were already free of micro- organisms and that particular care was taken in the aseptic manipulations, contaminants were introduced in one of the several steps involved in the complete procedure, i.e., rooting of the shoots, transfer and inoculation, 6 weeks incubation, removal of the plants, sectioning, and inoculation of the root pieces to culture media.

Plants of Alnus glutinosa propagated in vitro re- sponded in the same manner to the nodulation tests as the seedlings, i.e., each of the two clones used, AG-1 and AG-2, nodulated at 100% when inoculated with Fran- kia. These results confirmed those obtained by Lalonde et al. (1981) with axenic seedlings of A. glutinosa inoculated with pure cultures of Frankia AGNlg. The high nitrogen nutrition from plant tissue culture media did not prevent the subsequent nodulation of the plantlets propagated in vitro after their transfer to a nitrogen-free substrate.

he nodulation of axenic plantlets of A. glutinosa confirmed the fact that Frankia can induce typical and effective nodulation on axenic host plants without the help of any microorganism. Obviously in natural condi-

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PERINET AND LALONDE 2887

FIG. 1. Growth of nodulated Alnus glutinosa plantlet (left), inoculated with Frankia ARgN18f, compared with the uninoculated control (right) after 6 weeks. The Erlenmeyer flasks (experiment series 1) contained a mix of Vermiculite-Turface with N-free Crone's solution. Bar = 5 cm. FIG. 2. Actinorhizae formed on the roots of Alnus glutirlosa propagated in vitro and inoculated with Frankia A C N I ~ ~ in axenic conditions after 6 weeks of growth. Bar = 5 mm. FIG. 3. Growth of nodulated Alnus glutinosa plantlets after 6 weeks in culture tubes (35 X 200 mm) containing a mix of Vermiculite-Turface with N-free Crone's solution (experiment series 2). FIG. 4. Typical Frankia A C N I ~ ~ colonies growing on a root piece of Alnus glutinosa plantlet, after 3 weeks of incubation in the QmodB medium. Bar = 5 mm.

tions, rhizosphere microorganisms, such as the Pseudo- monas suggested by Knowlton and Dawson (1982), could interact in the infection process of the root hair by Frankia, particularly when the host plant is less recep- tive or if the environmental conditions, such as the pH, are unfavorable. Nevertheless, pure cultures of Frankia can nodulate at 100% aseptic plantlets of the Alnus glutinosa host plant when growth conditions are not extreme.

Acknowledgements We are indebted to P. Younger for reviewing the

manuscript.

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BAKER, D., J. G. TORREY, and G. H. KIDD. 1979. Isolation by

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