The influence of some common plant phenolics was tested on six Frankia strains isolated from both Alnus and Elaeagnus host plants. The addition of 1 mM of different phenolics to QmodB broth significantly influenced the growth and (or) the morphological development of Frankia. Ferulic, o-coumaric, and p-coumaric acids were extremely effective in inhibiting the growth of Frankia colonies, increasing the ramification of hyphae, and decreasing the number and size of sporangia produced in vitro. However, benzoic and p-hydroxybenzoic acids did not influence the total growth of Frankia colonies but stimulated the in vitro production of spherical septate vesicles on the two strains of Frankia type N tested. The possibility that these active phenolics, which are known to be present in Alnus actinorhizae, might act as "chemical mediators" between the host cell and its endophytic Frankia is suggested.
From 20 provenances of field-collected actinorhizae of Myrica gale, more than 3000 test tubes were inoculated with OsO 4 -treated nodules. Only 30 Frankia strains were isolated from 6 provenances. Most isolates showed an extremely slow growth in the various isolation media tested. For 12 strains whose growth was sufficient, the Frankia nature of isolates was verified by morphological characterization and biochemical analysis, using gas chromatography for the presence of 2-O-methyl-D-mannose. All 12 strains showed infectivity on M. gale and Alnus glutinosa. Five of those strains were ineffective on A. glutinosa but effective on Elaeagnus angustifolia and Hippophaë rhamnoides.
Caulogenic callus cultures were established from vegetative short shoot buds collected from cuttings from a ca. 12‐year‐old tree of Larix × eurolepis. Schenk and Hildebrandt basal medium supplemented with various concentrations of 6‐benzylaminopurine (BAP, 2.5 × 10 –7 to 1.0 × 10 –5 M) or BAP (5.0 × 10 –6 M) + indolebutyric acid (IBA, 1.0 × 10 –6 M) was used. Subcultures were carried out on the same medium as used for initiation. Shoot formation resulted mostly from adventitious organogenesis on callus tissue, but adventitious organogenesis and axillary shoot development were occasionally observed on neoformed shoots. Series initiated in December 1984, June 1985, and August 1985 were still very productive after 32, 26, and 24 months respectively in culture. Sampling date of the primary explants had a pronounced effect on i) initial survival rate, ii) number of subcultures before establishment of continuous, vigorous cultures, and iii) number of subcultures before attaining a high percentage of caulogenic calli and good productivity. Browning of callus tissue with time during each subculture was concomitant with an increase in shoot productivity. Most neoformed shoots did not show pronounced elongation of the stem in vitro. Some shoots entered dormancy, and either stayed dormant upon subculture or resumed growth. Depending on the concentration of growth regulators used during callus culture, between 4 and 22% of excised shoots rooted when transferred first on DCR medium without growth regulator (Gupta and Durzan, 1985) and then twice on DCR‐1 medium (Gupta and Durzan, 1985). Rooted shoots elongated after a few weeks in greenhouse conditions.
Vegetative tissues from 14 sexually mature populations of speckled alder (Alnus rugosa (Du Roi) Sprengel) in Québec have been analyzed for electrophoretically demonstrable variation in 9 enzymes encoding a total of 15 loci. Speckled alder demonstrated a diploid-like expression for all polymorphic loci investigated. Populations were in Hardy-Weinberg equilibrium for the 9 polymorphic loci observed. The total expected heterozygosity was 0.173. Analysis of fixation indices indicated a slight deficiency of heterozygotes, relative to panmictic expectations. This was likely due to weak population differentiation, which did not follow any particular geographical trend. Qualitative estimates of interpopulation rates of gene flow were high, and in good agreement with the small pair-wise population genetic distances and among-population fixation index. Levels of genetic diversity and partition of this diversity were similar to previous observations obtained with the sympatric species A. crispa (Ait.) Pursh. However, no phenomena of interspecific hybridization were noted. The average genetic distance between these two alder species was large, with a value of 0.4, emphasizing the different ecological niches colonized by each of the two species. The estimate of divergence time between these two taxa was two million years. It is in agreement with the hypothesis of repetitive spatial isolation of each species in ice-free refugia during the Pleistocene, thus promoting their reproductive isolation. The interspecific divergence noted at the enzyme level allowed for the easy electrophoretic identification of each taxon.
The inoculation of the European Alnus glutinosa (L) Gaertn host plant by a pure culture of an actinomycete isolated from the North American Comptonia peregrina (L) Coult root nodule was successful. Fluorescein-labeled antibodies, specific against the actinomycetal isolate of the C peregrina root nodule, demonstrated the identity of this actinomycete in the resulting Alnus root nodules The nodulation process of this abnormal host-endophyte system was studied by light and electron microscopy The actinomycetal intruder was branched, and septate hyphae produced septate vesicles These endophyte vesicles were spherical like the Alnus-type instead of club shaped like the Comptonia-type The endophyte hyphae and vesicles were always encapsulated in a polysaccharide material surrounded by a host plasma-membrane envelope The Comptonia isolate, which forms spores in pure culture, did not sporulate inside the Alnus root nodule. In this abnormal host-endophyte system, appearance of the root nodules was delayed for 1-2 wk, compared with a normal Alnus-endophyte system However, the delayed root nodules which developed on all of the inoculated Alnus seedlings were effective in fixing nitrogen and were able to support satisfactory plant growth in a nitrogen-free medium.
The inoculation of the European Alnus glutinosa (L.) Gaertn. host plant by a crushed-nodule inoculum, prepared with the North-American Alnus crispa var. mollis Fern, root nodule, was successful. Fluorescein- and ferritin-labelled antibodies, specific against the A. crispa var. mollis root nodule endophyte (Lalonde et al. 1975), demonstrated the identity of this endophyte in the resulting nodules. The nodulation process of this abnormal host–endophyte system was studied by light and electron microscopy. An excretion of host blebs containing electron-dense polysaccharide material, resulting in the formation of exo-encapsulation threads containing presumptive endophytic bacterial cells, was associated with deformed root hairs. Originating from an exoen-capsulation thread, the endophyte penetrates the root hair cell and then migrates as a hypha toward the cortical cells of the root. Its migration in the cortical cells of the primary nodule results in the induction of a lateral root which develops as the true nodule. The ultrastructure of the A. crispa var. mollis endophyte developing in the primary and true nodule of the abnormal A. glutinosa host was similar to the one induced inside its normal A. crispa var. mollis host. The actinomycetal intruder was a branched and septate hypha able to produce septate vesicles. The endophyte was always encapsulated in an electron-dense polysaccharide material surrounded by a host plasma membrane envelope. However, in this abnormal host–endophyte system, the number of primary nodules formed per root system was drastically reduced, and their appearance was delayed by 1 to 2 weeks. The delayed nodules were effective in fixing nitrogen and able to support satisfactory plant growth in a nitrogen-free medium.
