Confirmation of the infectivity of a free-living actinomycete isolated from Comptonia peregrina root nodules by immunological and ultrastructural studies

Abstract

The inoculation of the host plant Comptonia peregrina (L.) Coult. (Myrica asplenifolia L.) by a pure culture of a free-living actinomycete, isolated from Comptonia root nodules by Callaham et al. (1978, Science, 199: 899–902), was successful. Short-term and long-term nodulation tests confirmed the infectivity of the Comptonia isolate. Acetylene reduction assays of the nodules induced by this prokaryote isolate demonstrated nitrogen fixation activity. This nitrogen fixation activity was able to sustain a prolific growth of the nodulated host plants growing in a N-free substrate. Indirect immufluorescence reactions, using specific gamma globulin against the actinomycetal isolate of the Comptonia root nodule, demonstrated the identity of this actinomycete in these in vitro produced Comptonia root nodules. Ultrastructure of the Comptonia isolate, developing as a free-living or endophytic actinomycete, was observed by light microscopy, freeze etching, and transmission electron microscopy. The free-living filamentous and sporulating isolate produced typical hyphae and vesicles when growing as an endophyte in the Comptonia nodule. These endophytic hyphae and vesicles were always encapsulated by a polysaccharide material which was surrounded by a host membrane envelope. A polysaccharide capsule was not demonstrated on the free-living Comptonia isolate. The endophytic vesicles were club shaped and highly septate. Such vesicles were never produced by the free-living isolate growing in an artificial medium. The Comptonia isolate is a spore former in pure culture and was able to sporulate in basal tissues of 5-month-old Comptonia nodules.