Walls of the bacteroid form of Rhizobium leguminosarum, from plant nodules, and walls of free-living R. leguminosarum were isolated and compared by extraction with aqueous phenol and chemical analysis. Bacteroid walls had less lipopolysaccharide than walls isolated from bacteria cultured on four different media. This difference may be relevant to the relatively greater permeability of the outer membrane of bacteroids, and important in symbiotic interactions.
SUMMARYDuring incubation of nutrient solutions heavily inoculated with an axenic suspension of spore-nodules oi AInus glutinosa (L.) Vill. we observed an increase in infective capacity. This effect was only obtained with nutrient solutions to which an extract with petrol ethersoluble substances from roots had been added.The reproducibility of the experiments is definitely increased when the active lipid factor(s) is further purified by sihcagel column chromatography. Aerobic conditions are essential. Microscopic examination of the infective nutrient solutions showed the presence of Actinomycete-like hyphae. These hyphae were never observed in controls where the lipid extracts had been omitted. After several transfers to fresh media, cultures are still highly infective and microscopically characterized by growth of these hyphae.After inoculation with axenic suspensions of spore + nodules, neither an increase of infectivity nor growth of hyphae was observed.
INTRODUCTIONInterest in symbiotic nitrogen fixation by non-leguminous plants has increased during recent years (we refer to the recent reviews of ALLEN and ALLEN 1959and BOND 1958. The experiments done by Bond demonstrated the fixation of elementary nitrogen in root nodules of many non-legumes. It is, however, still impossible to cultivate the micro-organisms which are responsible for the formation of these nodules, even though they can be easily observed as endophytes in the nodular tissues. Many claims have been made that the endophyte of Alnus glutinosa had been isolated, but none of these claims has been able to stand the test of modern criticism. The only exception seems to be a very recent claim by POMMER (1959) that he had isolated an Actinomycete-like organism on plain glucose-asparagine-agar which was able to produce nodules on roots of Alnus plants under aseptic conditions. This claim by Pommer is so in contradiction to the experience of so many other investigators that his results are very difficult to reconcile with those of other experiments. We will try to do so after describing our own experiments.These experiments were based on the idea that a reproducible isolation of the endophyte of Alnus glutinosa or those of other nonleguminous root nodules will only be possible when the growth requirements of these endophytes are sufficiently known. One of the previous communications of this series (1956) described a method which makes it possible to determine the increase of the number of infective particles in a nutrient solution during incubation after inoculation with a small amount of a suspension of "pure" nodules. It was then possible to determine the increase in the number of infective particles during a three-week period of incubation at 27°C. in certain peat suspensions, although no increase could be observed in other peat suspensions. It was shown that this difference was due to the presence of growth-inhibiting substances in the latter peat
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