The Azolla, Anabaena Azollae Relationship
INITIAL CHARACTERIZATION OF THE ASSOCIATION1
Received for publication November 8, 1973 and in revised form JanLary 16, 1974
GERALD A. PETERS AND BERGER C. MAYNE Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387
Cultures of Azolla caroliniana Willd. free of the symbioticblue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source. Morphological aspects of the symbiotic association show the confinement of the Anabaena azollae within the leaf cavity of the Azolla. Procedures were established for the isolation of purepreparations of Anabaena azollae and Azolla chloroplasts. It has not yet been possible to grow the isolated alga in independent culture. Photochemical activities of the isolated alga and fern chloroplasts were measured by spectrophotometric assays for photosystems I and II as well as by P700-content (photosystem I) and delayed light emission (photosystem II). In the algal fraction, both photosystems wererepressed when compared to freeliving Anabaerna cylindrica, but the relative ratio of photosystem I to photosystem II may be appreciably greater in Anabaena azollae. Azolla chloroplasts were generally comparable to spinach chloroplasts. A comparison of the chlorophyll a and b content of Azolla fronds with and without the symbiotic alga resulted in an estimate that in the symbiotic association, theAnabaena azollae accounts for from 7.5 to 15% of the total chlorophyll.
26). Nickell (23), in securing an aseptic culture of Azolla
caroliniana, reported that both bacteria and algae were eliminated by treatment with antibiotics. Johnson et al. (18) used antibiotics after surface sterilization to remove green algae (largely Chlamydomonas) which continually appeared in cultures with combined N2.They stated that these treatments also eliminated Anabaena azollae from the cultures since the alga was not observed in the fronds. However, neither Nickell (23) nor Johnson et al. (18) demonstrated the absence of growth and N2 fixation in N2-free medium by the fronds in which the symbiotic alga was reportedly absent. The Azolla-Anabaena azollae association permits the study of a symbioticrelationship between a blue-green alga and a green plant under laboratory conditions. Previous studies on the physiology of the symbiotic association were not well defined and were limited in scope. Various aspects of mineral nutrition, temperature, and light intensity on the growth of the organism have been reported (22). We are not aware of any studies on the metabolic functions of N2 fixation,respiration, and photosynthesis in the individual organisms or their interaction in the symbiotic association. This manuscript is a report of initial studies on the characterization of the Azolla-Anabaena azollae symbiotic relationship. We describe the morphology, a method of freeing the fronds of the symbiotic alga, and isolation procedures devised to fractionate Azolla-Anabaena azollae for metabolicstudies. The companion publication deals with aspects of acetylene reduction (nitrogenase activity) (25).
MATERIALS AND METHODS Azolia Cultures. Plants of Azolla caroliniana Willd. were obtained from a local greenhouse pond. Cultures free of contaminating epiphytic microorganisms, especially green and blue-green algae, were obtained by washing fronds 20 times with vigorous agitation in largevolumes of distilled water, followed by surface sterilizing with a solution containing 2% Clorox (0.12% sodium hypochlorite) and 0.01% Triton X-100 as a wetting agent for 20 to 30 min. The fronds were removed, washed a minimum of six times with large volumes of sterile distilled water, and transferred to sterilized nutrient medium in Erlenmeyer or Fernbach flasks, with and without a combined N2...