L. Simon

Performance of in vitro propagated Alnus glutinosa (L.) Gaertn. clones inoculated with Frankiae

SummaryThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.

Confirmation of Frankia Species Using Cellular Fatty Acids Analysis

Summary The fatty acid methyl esters of 30 selected Frankia strains were analysed by capillary GLC. The temporal stability of the resulting profiles was demonstrated on a subset of strains. Multivariate statistics were used to analyse the data. The Frankia strains belonging to the species F. elaeagni and those of F. alni subsp. pommerii could be separated in different clusters. Furthermore, strains classified as F. alni subsp. vandijkii were shown to be outside the F. alni subsp. pommerii cluster. This was confirmed by reanalysing isozyme data available for the same strains. The clustering of strains achieved using fatty acid profiles thus corroborates the recently proposed taxonomy of Frankia .

Effect of substrate nitrogen on the performance ofin vitro propagatedAlnus glutinosa clones inoculated with Sp+ and Sp−Frankia strains

The addition of combined nitrogen to substrate at an appropriate rate can stimulate N2-fixation thus inreasing the efficiency of the Alnus-Frankia symbiosis. To examine how nitrogen additions can effect the peformance of different pairs of symbionts, growth and time course of N2-fixation were studied in plants supplied with NH4NO3. Two cloned ofAlnus glutinosa (L.) Gaertn., propagatedin vitro, were inoculated with two strains ofFrankia (AVP3d and ACN14a) and grown in a greenhouse. Calcined montmorillonite (TotfaiceR) was used as growth substrate. Six N treatments were made up of varied amounts of NH4NO3 supplied in one single addition shortly before inoculation. Weekly measurements of shoot height and repeated measurements of nitrogenase activity (acetylene reduction) performed on intact root systems were used to monitor the development of the symbioses.Nitrogen treatments containing from 0.10 to 0.68 mg N g−1 dry substrate stimulated N2-fixation as well as growth. The relative performance of the two clones was different according to N treatment; one clone showed a greater benefit from the nitrogen input. Our results support the recommendation that selection of symbionts according to performance should be carried out with an input of combined nitrogen. This can provide optimum conditions for the development of each pair of symbionts.

Fluorescently-primed in situ PCR in arbuscular mycorrhizas

In situ gene amplification (in situ PCR) is a recent, powerful molecular technique which allows the localization of low abundance nucleic acids targets directly within tissue sections. The work presented here is, to our knowledge, the first report of successful direct detection of in situ PCR amplification with fluorescently-labelled primers, and the first successful in situ PCR performed on arbuscular mycorrhizal (AM) fungi. Ribosomal SSU genes within AM fungal spore sections were amplified by using fluorescent, glomalean-specific primers, then directly detected by means of epifluorescence microscopy. Different controls confirmed the successfulness of the in situ amplification. These results open new avenues in the study of arbuscular mycorrhizas, where genetic processes seem to be transient and very localized.

Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages.The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO2 and plant flavonols.