Evaluation of Frankia strains isolated from provenances of two Alnus species

Evaluation of Frankia strains isolated from provenances of two Alnus species

Dipartement d'e'cologie et pidologie, Faculti de foresterie et ge'ode'sie, Universite' Laval, Qukbec, (Que'.), Canada GI K 7P4

Accepted June 15, 1982

NORMAND, P., and M. LALONDE. 1982. Evaluation of Frankia strains isolated from provenances of two Alnus species. Can. J . Microbiol. 28: 1 133-1 142.

Using the 0 s 0 4 isolation method, more than 200 Frankia strains were obtained from 27 provenances of the two alder species represented in Quebec, i.e., Alnus crispa (Ait.) Pursh. and Alnus rugosa (Du Roi) Spreng. The Frankia isolates were evaluated for morphological characteristics, infectivity, and efficiency. Variations in these factors were noted between provenances and also between isolates from a single provenance. The distribution of sporulating (Sp') and nonsporulating (Sp-) type isolates was found to be related to host plants and provenance. The sporulating or nonsporulating endophytic character was found to significantly affect efficiency. This endophytic character was recognized as one of the valid criteria that should be used in the awaited species definition in the genus Frankia.

NORMAND, P., et M. LALONDE. 1982. Evaluation of Frankia strains isolated from provenances of two Alnus species. Can. J . Microbiol. 28: 1133-1 142.

, Plus de 200 souches de Frankia ont ktk obtenues Zi partir de 27 provenances des deux espkces d'aulne (Alnus crispa (Ait.) Pursh. et Alnus rugosa (Du Roi) Spreng.) indigknes au QuCbec, p i c e Zi l'utilisation de la mkthode d'isolement au OsO,.

; L'Cvaluation de ces souches a kt6 baske sur les caractkristiques morphologiques, I'infectivitk et I'efficacitk. Ces facteurs varient i entre les souches d'une provenance et entre les provenances. La distribution des souches de type sporulant (Sp+) et non-sporulant I (Sp-) est relike aux plantes-hates et aux provenances. Le caractkre sporulant ou non-sporulant de l'endophyte est relik Zi une i diffkrence d'efficacitk. Ce caractkre de l'endophyte est reconnu comme un des critkres valides Zi utiliser dans la dkfinition 1 attendue des esp&ces du genre Frankia.

1 I Introduction Callaham et al. (1978) obtained a pure culture using an ! ~ i ~ l ~ ~ i ~ a l nitrogen fixation, important on a global elaborate and time-consuming enzymatic method. Since ! basis, is essential to the development of certain gosys- then, four methods have been used that have ~ielded : (memans and ~ ~ ~ l ~ f ~ ~ ~ 1980). ~h~ necessary pure Frankia cultures: sucrose density gradient centrif- ) infomation for that function is only present in the ugation (Baker et al. 19791, serial dilution (Lalonde

genotype of some prokaryotes (Ruvkun and Ausubel 1979a)9 selective incubation (Quispel and Burggraaf 1980), two of which have established an endophytic 198 1 ) ~ and osmium tetroxide ( 0 ~ 0 4 ) matment (Lalonde

symbiosis with extensively represented groups of plants: et 98 and variations Of these. The 0s04 method : ~ h i ~ ~ b i ~ ~ with the legumes and ~ ~ ~ ~ k i ~ with the was chosen because it is rapid, easy to use, and reliable.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... :.. .: . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .: -..: ..; ... : .... .;:..: :, actinorhizal plants. Fifteen genera of pioneer plants because it can yield many from One . . . . . . . . . : . . . .:. ':- :..:..::: .....,...'] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . are now known to possess actinorhizae or Frankia- lobe, one can the genetic existing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . I containing nitrogen-fixing root nodules ( m e m a n s and within a provenance¶ as as between. . . * . . . . . . . ,! . . . . . . .

., . : H~~~~~~ 1979). ,qnus, a subtropical to subarctic The assessment of the extent of variation in biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . genus, has two species represented in Q ~ ~ ~ ~ ~ , speckled accretion and in infectivity is of foremost importance in

. . alder (Ainus rugosa) and green alder (A. crispa). Both a program of Of the Therefore, , species have been considered for short rotation biomass a large-scale isolation and evaluation of Frankia strains

production on poor soils ( ~ ~ ~ i ~ et al. 1979) and this from different provenances was planned. The in vitro , requires a comparative itiidy ofthe relative effidiency of morphology of isolates of the so-called spore-forming

different Frankia strains. (Sp') endophytes (Van Dijk and Merkus 1976) and their pure cultures of ~ ~ ~ ~ k i ~ are necessary for evaluation efficiencies in fixing nitrogen were studied. The distri-

in order to standardize the trials and eliminate the bution pattern in Quebec of the S ~ - - and S ~ + - t ~ ~ e influence of the compounds present in the so-called Frankiae was also investigated in relation to the host "crushed-nodule inocula" that were used previously to plants and their provenances- inoculate actinorhizal seedlings. But before 1978, no pure culture was available for such studies. Since the Materials and methods turn of the century, some 40 teams had attempted the Sign$cance of acronyms isolation of Frankia (Baker and Torrey 1979) before The Frankia isolates were given an acronym according to

0008-4166/82/101133-10$01 .OO/O 01982 National Research Council of Canada/Conseil national de recherches du Canada

. . . . . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

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1134 CAN. 1. MICROBK

the system of Lalonde et al. (198 1). First, one letter or a group of letters to describe the host species (e.g., AC for A. crispa, ARg for A. rugosa, T for isolates obtained from growth pouch cultivated seedlings inoculated with Frankia-containing soils), then a letter to define whether or not sporangia were observed in the nodules from which the isolation was performed (e.g., ACN7a is an isolate obtained from a nonsporulating endophyte and ARgN/P18 is a provenance from which both types of isolates were obtained). Then comes an arbitrary number given to each provenance and a letter to identify the different isolates from one provenance (ACN14a, ACN14b, .... ACN14j). Then, at the upper right comer are sometimes places letters to describe the fact that the strain was reisolated from a different host species (ACN8aAG is an ACN8a isolate obtained initially from A. crispa and then reisolated from A. glutinosa). When those letters are under- lined, it means that the host plant was inoculated with a crushed-nodule inoculum (Lalonde 1979a) and the the isolation was then performed without a previous direct isolation (ARgp5A-G).

T N l 5 a K for instance is the isolate a from provenance 15, from which a dried soil sample (T) was inoculated to A. crispa (AC) and found to produce Sp- nodules (N).

Microscopic observation of nodule sections To determine the endophyte strain type (Sp- or Sp'),

nodule lobes were excised, and 12-pm-thick sections obtained from a Hooker plant microtome (Labline Instruments Inc., Melrose Park, IL) were stained with cotton blue and observed with the X40 and XlOO objectives using the bright-field (Lalonde 1979b) and interference-contrast optics (Ortholux 11, Leitz). That technique was also used to confirm the strain type on the pouch and styroblock seedlings inoculated with the isolates.

Isolation and growth of Frankia Nodules from which isolation was performed were obtained

according to three different experimental procedures: first, greenhouse alders, from surface-sterilized seeds, planted in Turface (International Minerals and Chemicals Corp., Des Plaines, IL), fertilized with N-free Crone solution (Lalonde 1979b), and inoculated with Frankia-containing soils from the field; second, uprooted plants from the field, transplanted in pots containing Turface, and kept in the greenhouse; or third, directly from nodule clusters excised in situ and treated within a few hours. Each provenance consisted of the nodule clusters collected from one plant.

Water-cleaned and paper-blotted nodule lobes were im- mersed in a 3% (w/v) aqueous 0 s 0 4 solution for periods ranging from 5 s to 6 min (Lalonde et al. 198 1). After three successive rinsings in sterile distilled water and 5-15 min of incubation in sterile PVP-PBS (polyvinylpyrrolidone, molecular weight 40000, soluble (Sigma, St. Louis, MO) and phosphate-buffered saline: Na2HP04.7H20, 2.16 g.L-'; KH2P04, 0.2g. L-'; NaCI, 0 .8g .~-1) solutions at 0.1% (w/v) PVP concentration for A. crispa and 3% (w /v) for A. rugosa, the single lobes were crushed in new sterile PVP-PBS solutions and transferred to 20 x 150mm tubes containing 14 mL of Qmod B medium (Lalonde and Calvert 1979) with 5 mg.L-' L-a-lecithin (Sigma, St. Louis, MO, catalogue No. P5638). A total of 50 tubes was normally used for each provenance of nodules and these were incubated at 27°C.

IL. VOL. 28. 1982

After the emergence of fluffy whitish outgrowths on the blackened surface of the nodules, tubes were observed with the naked eye, and the microorganism present was observed by microscopy using the X 100 objective of the phase- or interference-contrast optics to make a presumptive identification of the isolates as Frankia (Lalonde et al. 1981).

Subculture Ten tubes containing pure presumptive Frankia isolates

were chosen at random and sealed with a rubber serum cap, and the colonies were broken with a syringe and needle (20 gauge). For the first subculture, four fresh Qmod B tubes were inoculated. The dilution factor, low at first (1 in 4), could be increased up to 1 in 50 or more upon the second subculturing in the case of Sp--type isolates. For some Sp+-type isolates from A. rugosa, a 1 to 1 subculturing had to be used. The top-layer agar method from Quispel and Burggraaf ( 198 1) was tried for subculturing the Sp+-type isolates from A. rugosa: a bottom 1% (w/v) agar layer containing Qmod medium without peptone was covered with a 0.8% (w/v) agar layer containing peptone and 5 m g . ~ - ' lecithin, cooled to 40°C, and inoculated with the fragmented colonies of presumptive Sp' Frankia isolates. The Petri dishes were sealed with masking tape (3M, London, Ont.) to prevent dehydration and incubated at 27°C. Standard method agar (SMA) (Baltimore Biological Labora- tory, Inc., Baltimore, MD) plates and Qmod B tubes supple- mented with 7% (v/v) fetal bovine serum (FBS) (GIBCO, Grand Island, NY), whose purpose was to inhibit sporulation (Lalonde and Calvert 1979), were also used for subculturing A. rugosa Sp+-type presumptive Frankia isolates.

Infectivity test Infectivity tests in growth pouches (Scientific Products,

Evanston, IL) were done according to Lalonde (1979b). The A. glutinosa seeds were surface- sterilized by mechanical agitation in 30% H202 with a trace of Tween 20 (Sigma, St. Louis, MO) as surfactant for 5-20 min. Five to 10 seeds were placed in the trough of each growth pouch to which 15 mL of N-free Crone solution had been added. The pouches were placed in growth chambers at 15 000 Ix for 15 h, 22°C day/l7"C night, for germination and growth. The seedlings were inoculated any time after the emergence of root hairs by placing a drop of PBS-washed and fragmented Frankia isolate on the roots. The pouches were fertilized bimonthly with 10 mL of N-free Crone solution.

Ejficiency test The efficiency tests in styroblocks were done according to

Lalonde and Calvert (1979). The A. crispa seeds from Val d'Or (48" N, 78" W) were soaked in 2°C water for 3-4 days and surface sterilized as described above. The seeds were placed four or five per Turface-filled cavity of No. 8 styroblocks (Mansonville Plastics, Montreal, P.Q.) previously fertilized with N-free Crone solution and germinated under transparent plastic with the same growth chamber conditions used for growth pouches. After approximately 1 month, the N-starved seedlings were inoculated with a I-month-old isolate colony cluster from one tube for each 15 cavities. The isolates were washed in PBS and fragmented with a 20-gauge 1-in. (25.4 mm) needle and a 3-mL plastic syringe. Five uninoculated controls were kept for each isolate tested. After 3 months of growth, the shoot height, fresh weight, oven dry weight,

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NORMAND AND LALONDE

TABLE 1. List and geographical origin of isolates*

Coordinates Soil Site

Acronym Isolate letters Location tYPe type N W Source?

ACN3 ACN4 ACN5 ACN6 ACN7 ACN8 ACN 10 ACNll ACN12 ACN13 ACN14 TNISK ACP 17 TN 1 8 K ACN20 ACN2 1 ARgN4 ARgp5G ARgP6 ARgN8 ARgN9 ARgP12 ARgN14 ARgN/P18

ARgN22 ARgN25 ARgN30

Tadoussac Cap-aux-Oies Lac Beauport Gasp6 Perc6 Baie-St-Paul Tadoussac Baie St-Paul Petite Rivikre Petite Rivikre Tadoussac Ste-Monique Breakeyville Manitounuk Ste-Louise-des-Aulnaies Port-au-Persil Pointe-i-la-Garde Quebec City Tadoussac S herbrooke St-Calixte St-Samuel St-Pierre

Grand-M&re St-Magloire St-Omer Albanel

*The acronym of the provenances (Materials and methods), the isolates letter (in the case of ARgP/Nl8, the Sp type is given in parentheses), the name of the locality or municipality, the soil type as determined by hand (SS, sandy soil; SL, sandy loam; CL, clay loam), the site type (RS, road side; W, wild; DA, disturbed area; DD, drainage ditch), and the coordinates (latitude and longitude) are given.

TPY, Patti Younger; YP, Yves Perradin; AF, Dr. I . Andr.4 Fortin; LS, Louis St-Laurent; JD, Dr. Jeff Dawson; A, authors.

and N concentration (micro-Kjeldahl) (Coles and Parks 1946) were measured.

. . Acetylene reduction assay . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . : The 3-month-old seedlings from the infectivity tests were . . . . . . . . . . . . . . . . . . . . . . - - - . analysed for acetylene reduction and ethylene production

according to Koch and Evans (1966). The root systems were . .

washed under running water, and 0.1 g fresh weight of nodules was excised and introduced into a 20 x 150 mm glass test tube sealed with a rubber serum cap. Ten percent of the atmosphere was then replaced with acetylene and the tube was incubated for 1 h at room temperature. Controls without acetylene were also tested. Using a gastight glass syringe, a 25-ILL sample was injected into a gas chromatograph (Hewlett-Packard, model HP5710A) equipped with a flame ionization detector and a Porapak N (80-100 mesh) filled column (6 ft (1.8 m) X Q in. outside diameter X 2 mm inside diameter). The injection port, column, and detector temperatures were 100, 60, and 120°C, respectively, and the flow rates of Hz, air, and N2 carrier gas were 20, 200, and 30 mL-rnin-', respectively.

Lyophilization The 3- to 5-week-old colonies were transferred with

approximately 0.5 mL of the original Qmod B medium into 2-mL prescored ampoules (Wheaton Sc., Millville, NJ). The ampoules were frozen at -85'C, desiccated in a Virtis freeze- dryer for 3 h, and vacuum sealed. The validity of this technique was verified by checking survival of the Frankia cells in subcultures of the freeze-dried samples in tubes of new Qmod B medium.

Results Isolation of Frankia strains of both A . crispa and A.

rugosa endophytes was always successful when sound nodules from healthy looking plants were used.

Isolations In less than a year, 220 isolates were obtained from 27

provenances of A . crispa and A. rugosa (Table 1 ) . Following the 0 s 0 4 treatment, a whitish outgrowth occurred on the surface of the blackened nodule epider- mis after an average of 2 weeks for Sp--type and 4 weeks for Sp+-type nodules. In the case of A. rugosa Sp+-type nodules, this adaptation phase could last up to

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CAN. J. MICROBIOL. VOL. 28, 1982

6 weeks. Typically, of the 50 tubes inoculated with crushed nodule lobes from one provenance, from 0 to 50 tubes with an average of 10 were contaminated by bacteria, actinomycetes, yeasts, or fungi. Of the remain- ing uncontaminated tubes, from 0 to 100% with an average of 80% (-30 tubes) were found to contain presumptive Frankia isolates. This identification was based on the observation under the interference light microscope of typical Frankia features, i.e., septate and ramified hyphae, sporangia, and spherical vesicles.

Subculture and growth For Sp--type nodule lobes, 1 month after 0 s 0 4

treatment the sealed tubes were subcultured by breaking the colony clusters and injecting them into four fresh Qmod B tubes (a 1 in 4 dilution ratio). On the second subculturing, an acceleration in growth presumably occurred, coupled with an adaptation of the isolate to its new in vitro growth conditions, from its former endophytic to its present free-living state. Colony clusters that were ready for subculturing or for infection in efficiency and infectivity tests were obtained in 10 days. In the case of some Sp-- and Sp+-type isolates from A. crispa (ACN8, ACP17) and of all Sp--type isolates from A. rugosa (ARgP6, ARgP18), the time period required for the emergence of an outgrowth large enough for subculture (when the whitish mat totally covered the nodule lobe) was 2 months, and in the case of the Sp+-type isolates from A. rugosa, the dilution ratio also had to be reduced to 1 in 2. With some isolates from A. rugosa, growth did not resume and the isolates remained dormant. The ACP17 and ACN8 isolates showed a much better growth rate than the A. rugosa Sp+-type isolates that did not resume growth in standard Qmod B tubes. The isolates ARgP6b and ARgP6d were reisolated from A. crispa ( A R ~ P ~ ~ ~ ' and ARgp6dAC) to check the stability of the slow growth rate character, and no growth rate increase was noted: a whitish mat grew o n the surface but upon subculturing at a 1 in 4 dilution ratio, only very tiny (1 mm) and rare colonies (two to three per tube) occurred after 2 months.

~ re sum~t ive Frankia isolates were obtained from field-grown plant nodule clusters that were treated directly, until after leaf fall in November. No difference in time delay necessary for emergence of the Frankia colonies, nor any difference in in vitro growth rate or morphology, was noted among spring, summer, or fall isolates.

The ARgp5@ isolate obtained by the serial dilution technique (Lalonde 1979a) required 2 months for the appearance of colony clusters in Qmod B medium. When reisolated by the 0 s 0 4 technique from A. crispa grown in growth pouches (ARgP5A--G AC) the time required for appearance of colony clusters was 2 weeks.

Morphology The morphology of the isolates was in general as

follows: a whitish mat covering the bottom of the tube; under the light microscope X 100 objective, slender (0.5-1.Opm in diameter), branched, and septate hyphae, sporangia of various shapes and sizes (5- 100 pm in diameter) enclosing 0.2- to 1-pm-diameter spores; and occasional spherical and septate vesicles (2-3 pm). The isolates of Spt-type endophytes produced much denser colonies to the point where the colonies had to be disrupted before any observation under the light microscope could be made. The Sp+-type isolates also produced more numerous and bigger sporangia (up to 150 pm in diameter) as compared with the usual 20- to 50-pm-diameter sporangia of Sp--type sporangia. Some isolates (ACN8 and ARgN25) did not form normal sporangia but only very rare tiny (5 pm) outgrowths that appeared to be aborted sporangia.

One of these strains (ACN8a) was reisolated after inoculation to A. glutinosa ( A C N ~ ~ ~ ~ ) to check the genetic stability of this character, and no increase in sporangia number or size was noted between the two isolates. A summary of the differences between Sp+- and Sp--type Frankia is given in Table 2.

Infectivity Of the 220 isolates tested, all were found infective,

forming prenodules in 8-10 days after inoculation and, 1 week later, nodules that permitted the A. glutinosa hosts to grow in N-free Crone solution, as attested by green leaves and height compared with the stunted chlorotic uninoculated controls. The cotton blue stained nodule sections showed the same morphology as the field-grown nodules described above.

Microscopic observation of nodule sections In the cotton blue stained nodule sections, we

observed Frankia hyphae and vesicles growing in the midcortical cells. Hyphae invaded the apical cells, forming a central network, and later, vesicles developed on the periphery of the cells. In some provenances, we observed intercellular and intracellular sporangia (Sp'). The location of the Sp--type provenances was generally in the St. Lawrence lowlands, which are predominantly colonized by A. rugosa. In the highlands where A. crispa is dominant, all A. crispa nodules were of the Sp- type, and the A. rugosa host, growing in its atypical dry upland habitat, generally had Sp--type nodules (ARgN14, 22, 25) except for some Sp+-type nodules (ARgP6). In the introgression zone, i.e., in the valley north of 47" N, and in the mountains south of 47" N, both Sp+- and Sp--type nodules were observed on A. crispa and A. rugosa host plants (Fig. 1).

Acetylene reduction assay All nodules formed by 10 different isolates on the A.

glutinosa seedlings showed acetylene reduction (ethylene production) in the range of 0.5-15 pmol ethylene-h-' -g oven-dry weight of nodules-'.

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TABLE 2. Differences between Sp+- and Sp--type isolates

Character (terrain) SP+ SP-

Endophy te Hyphae, vesicles No difference Sporangia Numerous Rare Predominant host A . rugosa A. crispa Predominant habitat Wet lowlands Dry uplands

Isolate Hyphae, vesicles No difference Sporangia Numerous Less numerous Size Large (up to 150 pm) Small (less than 50 pm)

Growth rate Colony density

Slow Rapid High (compact) Low (fluffy)

Endophyte (infection by isolates on young seedlings)

Hyphae, vesicles No difference Sporangia Few None Infectivity No difference Efficiency Low (70% of that of Sp-) High

FIG. 1. Map of southern Quebec showing the main water courses (dark), the St. Lawrence and Saguenay floodplains (white), and the above 500-ft highlands (light gray). R, provenances of A. rugosa isolates; C, provenances of A. crispa isolates; T, isolates from soil-inoculated A. crispa seedlings. Superscripts: + , isolates were of the Sp+ type; + /- , both Sp- and Sp+ types were isolated. The hatched line represents the approximate southern limit of widespread occurrence of A. crispa on both sides of the river.

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1138 CAN. J . MICROBIOL. VOL. 28. 1982

FIG. 2. The seedlings of A. crispa grown in styroblocks inoculated with ACN8d, ARgP6b, and ACNlOt Frankia isolates showing differences in seedling height. The center styroblock was inoculated with an Sp+-type isolate and the other two with Sp--type isolates obtained from two different provenances.

Lyophilization All the isolates which could be subcultured in

Qmod B tubes were lyophilized in three replicates. Some strains such as A R ~ P S E , ACN8a, ACN-lOa, ACN14a, etc., which held a particular interest were lyophilized in larger quantities. The A. rugosa Sp+-type isolates such as ARgP6, ARgP12, and ARgP18 which had a poor growth were not lyophilized but were kept as "liquid culture" in rubber serum capped tubes and deposited in the collection.

EfJiciency From the 220 Frankia isolates obtained, a total of 36

were assayed for efficiency in three successive tests in styroblocks (Fig. 2). The parameters measured (dry weight, fresh weight, height, and N content) were found to be well correlated (e.g., R. = 0.94 between height and N content averages of isolates). The three tests were conducted under different growth conditions; relative humidity, light intensity, and temperature were not consistent throughout. Also, the time lapse between seedlings establishment and inoculation varied; therefore, the overall averages of height and N content for each test were different (Fig. 3). To make the tests comparable, the data were transformed into percentages of the most effective in each test.

The percentages were then linearized by the formula

arcsin fi, and a Bartlett test showed that the variances of the Sp+- and Sp--type isolates averages were homogenous. Thus, a Student t-test (Steel and Tome

N CONTENT vs HEIGHT ( raw data 1

Height (cm 1 FIG. 3. N content versus height illustrating the variation

between the two parameters. The plotted numbers refer to the three successive tests (Nos. 1, 2, and 3) and are averages of each isolate. The circled 1,2, and 3 are the overall averages of each test to show the difference caused by different growth conditions. The circled C is the average of all uninoculated controls.

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i NORMAND AND LALONDE 1139

N CONTENT vs. HEIGHT ( transformed data

Height ( % of max.)

FIG. 4. N content versus height o f the shoots, both expressed as a percentage o f the highest in each efficiency test. +, Sp+-type isolates; -, Sp--type isolates; double box, ACN8

' isolates; circled minus, Sp- isolates which had an efficiency comparable with that o f Sp+-type isolates (superscripts: a , ARgNl8d; b , ACNl lb; c , ARgN14a; d , ~ ~ 1 5 d s ) .

1960) was permissible; when done, it showed that the Sp+- and Sp--type overall averages were significantly different at the 99% level for both the N content and height averages. For these reasons, it was found that the two isolate types could best be represented as two overlapping sets or clusters on a graph of N content and

height averages (percentages) (Fig. 4). The Sp- isolates ACN8, ARGN18a, ARgN14a, ACNllb, and ~ ~ 1 5 d s were found within the Sp+ cluster.

The variations in efficiency, as measured by seedling height, within and between provenances were generally not significant except for ARgP6, ARgN14, and ARgN 18 provenances (Table 3). Isolates from provenances ACN10, ACN11, ACN8, ACN13, ACN14, ARgN22, T N ~ S C , and ACP17 were not significantly different. It is also shown in Table 3 that the most effective isolates were always of the Sp- type (ACN10, ACN14, and ARgN14) whereas the Sp+-type isolates (AR~PSG, ARgP6, and ACP17) had an efficiency that was on the average 70% of that of the most effective Sp--type isolates. The host origin of the isolates was not a factor, since in test No. 3 an A. rugosa strain was most effective and an A. crispa strain was least effective, a situation contrary to that of test No. 2. The difference in height between A. crispa seedlings inoculated with a Sp--type isolate (ACNlO), a Sp+-type isolate .(ARgP6b), and the atypical ACN8 in vitro nonsporulating isolate is illustrated in Fig. 2.

Discussion The morphology of the Frankia isolates was similar

to that of the isolates described by Callaham et al. (1978), Berry and Torrey (1979), Lechevalier and Lechevalier (1 979), Baker and Torrey (1 979), Lalonde

TABLE 3. Height o f seedlings in efficiency tests*

Test No. 1 Test No. 2 Test No. 3

Isolate Height? Isolate Height? Isolate Height?

ACNlOa ACNlOb ACNlOc ACNlOd

ACNl la ACNll b

Control 1.520.5 d ACNl lm 21.1k4.6 a

ACNlOt 21.9k3.9 a

ACN8d 17.524.5 b

ARgP6b 13.9k6.1 c ARgP6d 18.4k3.8 b

ArgN14a ARgN14b

ARgN22a ARgN22d

ARgN18d ARgN 18 f

T N ~ s ~ A - - C TN15d

ACP17a ACP17b

11.123.3 bcd 14.2k3.3 a

12.9k2.5 ab 12.122.6 abc

10.122.0 cd 12.7k3.1 ab

12.6k2.7 ab 11.122.7 bcd

9.822.0 d 9.1k2.3 d

'Results from the efficiency tests in styroblocks. ?Mean height ? SD. Within each test, values followed by the same letter(s) are not significantly different at the 95%

level.

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1140 CAN. J. MICROBI( 3L. VOL. 28, 1982

and Calvert (1979), and Lalonde et al. (1981), except that typical Frankia vesicles (Lalonde and Calvert 1979) were found in all isolates.

The isolates obtained were all infective, forming nodules as described by Angulo Carmona (1974). The nodule morphology was similar to that of original isolations of Frankia, thereby fulfilling Koch's postu- lates. This fact, that all isolates were infective, is to be compared with the situation for other collections of isolates. For instance, the two Frankia isolates from Casuarina nodules described by Gauthier et al. (1981) were not found to be infective. The isolates obtained by Callaham et al . (1978), Berry and Torrey (1979), Baker and Torrey (1980), and Quispel and Burggraaf (1981) were all infective, although one isolate from Elaeagnus was definitely classified as noninfective (Baker et al. 1980) and some isolates listed as Streptomyces may have been noninfective and in vitro nonsporulating Frankia (Berry and Torrey 1979).

It is possible that disruptive isolation techniques such as those used by Gauthier et al . (1981) and Baker et al . (1980), by destroying most of the Frankia material present in the nodule, caused not only a much longer adaptation period, but also obliged the cells to undergo more divisions to attain a number sufficient for subculturing than with the 0 s 0 4 technique. A higher division number means an increased possibility of mutations such as nonnodulation.

The morphology of the nodular endophyte of host plants A. crispa and A. rugosa was similar to that described in Lalonde and Fortin (1973) and Lalonde and Knowles (1975). The presence of spores and sporangia (Van Dijk and Merkus 1976) was noted, but contrary to Kiippel and Wartenberg (1958) and Van Dijk (1978) who could not discern any relationship between the presence or absence of sporangia and ecological factors, we found a pattern of distribution in which Sp--type endophytes were predominantly associated with A. rugosa in the fertile lowlands and Sp- with A. crispa in the dry uplands. We therefore suggest that the situation found in Quebec could throw light on the ecological significance of the two-types concept (Van Dijk 1978).

The Zweek time required for the appearance of presumptive Frankia isolates after isolation with the 0 s 0 4 method contrasts with the Zmonth period when other more disruptive techniques are used such as the serial dilution used for A R ~ P ~ ~ , the sucrose density centrifugation (Baker and Torrey 1979), or the selective incubation (Quispel and Burggraaf 1981). The isolation procedure that we used, i.e., the 0 s 0 4 method (Lalonde et al. 1981), was always successful with all Sp--type endophytes, but the growth and subculture conditions used for the Sp+-type isolates were less adequate. The phenols present in greater amounts in A. rugosa nodules and the difference in growth requirements of Sp+-type

isolates according to host plant origin (AC versus ARg) were probably responsible. The in vitro growth rate of ACP17 isolates was lower than that of the other A. crispa Sp--type isolates, but was higher than that of the A. rugosa Sp+-type isolates, a finding similar to that of Burggraaf et al . (1981) who found that Myrica gale Sp+-type isolates had less strict growth requirements than A. glutinosa Sp+-type isolates. The fact that ARgP6b did not grow faster after reisolation from A. crispa ( A R ~ P ~ ~ * ' ) may mean either that a genetic determinant is involved or that it is a quite stable phenotypic character. This is often the case with actinomycetes whose morphology is influenced f o ~ many subcultures by the previous history of the culture (Waksman 1959).

The efficiency tests showed that generally no significant differences existed between isolates from one provenance. The exceptions were three A. rugosa provenances (ARgP6, ARgN 14, and ARgN 1 8).

The variations between provenances were significanl in some cases but the most important source of difference was the Sp+-type and the Sp--type endophytic character (Fig. 4). The most effective inoculant as determined by the biomass assay in the three tests was oj the Sp- type (ACN10, ACN 14, and ARgN 14) and the least effective in two tests out of three was of the Spt type from both hosts plants (ACP17 and ARgP6). ARgp5g was better than the ACN8 isolates whick appeared to be natural variants or mutants that did no1 sporulate in vitro and had a consistently lower efficiencj as well as a slower in vitro growth rate. They appeared tc be degenerate Sp+-type isolates. The Sp--type isolate: which fell in the Sp+ cluster do not appear to be norma Sp--type isolates, since there is a gap in the distributior of efficiencies (Fig. 4). But the facts that no Sp+-type isolate was found in the Sp- cluster and that the large majority of Sp--type isolates formed a dense clouc validate the definition of two clusters which, further. more, were found to be significantly different at the 99% level. The fact that few Sp+-type isolates were testec does not refute the definition of two clusters, since thir was due to the fact that Sp+-type Frankia strains art harder than Sp--type strains to isolate and still harder tc grow in pure culture.

The existence of a significant efficiency differenct between the Sp+-type and the Sp--type isolates hac been suggested by Hall et al. (1979) who used crushed nodule inocula of Sp+ and Sp- types on A. rubra and A glutinosa. C. A. Maynard (1980. Ph.D. Thesis, low: State University, Arnes, IA, U.S.A.) also found such 2

difference between Sp+ and Sp- crushed-nodule inoc ula on A. glutinosa, as well as a marked effect of thc inoculum previous history on efficiency.

The Sp+- and Sp--type character and the previou: history of the isolate would then be the most importan

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NORMAND AND LALONDE 1141

source of variation between strains. These major differences due to the Sp'- and Sp--type character and the minor differences noted between isolates of one provenance and between provenances validate our procedure of isolating up to 10 isolates from each provenance and from many provenances, a procedure which has been made p ~ ~ ~ i b i e by the 0 s 0 4 isolation technique (Lalonde e ta l . 1981).

We suggest that due to the observed endophvtic (in vitro) morphological differences (Van Dijk i9?8), 'in vitro differences in rate of growth (Quispel and Bur- ggraaf 1981) and in morphology (Table 2), and the

1 significant differences in efficiency between Sp- and . . . . . . . . . . . . : Sp' types (Fig. 4), the "spore type" character should be

. . . . . . . . . . . . . . . . . . . . . . . - . . . - - , ; recognized as a valid criterion to be used as one of many

. . . .,

- - ' in the future species definition in the genus Frankia. It is

also recommended that the "Sp+" and "Sp-" terms refemng exclusively to the endophytic stage of Frankia should be replaced by the expressions "type P (posi- tive) and "type N" (negative) to designate, respectively, "Sp'" and "Sp-" in both their endophytic and free- living stages. The acronym system of Lalonde et al. (1981) using N and P has already been used for both endophytic and free-living Frankiae.

I Acknowledaements -

Thanks are expressed to Dr. C. Camiri for help with the Kjeldahl analyses and to Dr. R. Letarte for lyophilization of the isolates. This study was made possible by a Formation de chercheurs et d'action concertie scholar- ship to P.N. and a Natural Sciences and Engineering Research Council of Canada grant (No. 7 192) to M.L.

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Documents

Evaluation of Frankia strains isolated from provenances of two Alnus species