Isolation, Culture, and Behavior of Frankia Strain HFPCgI4 from Root Nodules of Casuarina glauca Author(s): Samira R. Mansour, Ahmed Dewedar, John G. Torrey Reviewed work(s): Source: Botanical Gazette, Vol. 151, No. 4 (Dec., 1990), pp. 490-496 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/2995335 . Accessed: 03/04/2012 15:56 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. The University of Chicago Press is collaborating with JSTOR to digitize, preserve and extend access to Botanical Gazette. http://www.jstor.org BOT. GAZ.151(4):490-496. 1990. (C)1990 by The Universityof Chicago. All rightsreserved. 0006-8071 /90/5 104-00 1 5$02 .00 ISOLATION,CULTURE, AND BEHAVIOROF FRANKIASTRAIN HFPCgI4 F1tOMROOT NODULES OF CASUARINAGLAUCA SAMIRAR. MANSOUR,'AHMED DEWEDAR,' AND JOHNG. TORREY HarvardForest, HarvardUniversity, Petersham, Massachusetts 01366 Casuarina glauca (Casuarinaceae)is an importantintroduced tree species in Egypt, valued for wind- breaks,land stabilization,and soil improvementassociated with actinomycete-inducedroot nodulesthat fix atmosphericnitrogen. A strainof Frankia designatedHFPCgI4 was isolatedfrom root nodulescollected in Egypt and its characteristicsassessed both in pure cultureand in symbiosis. StrainCgI4 grows well in syntheticnutrient medium with addedNH4+ or, in the absenceof combinedN in the medium,forms vesicles andfixes dinitrogenadequate for growth.Hyphae, vesicles, sporangia,and spores characteristic of the genus Frankia were observed.This strainshows spontaneousspore release when grown in medialacking N. When tested for infectivityon actinorhizalhost plantsgrown in unaeratedwater culture, CgI4 nodulatesseveral species of Casuarina that fix atmosphericdinitrogen. Other genera in the Casuarinaceae,namely, Alloca- suarina and Gymnostoma, were not nodulatedunder these conditions.Species of the genus Myrica and Comptonia peregrina (Myricaceae)were effectively nodulatedby CgI4. The isolate may have use as an inoculantfor forest plantationsusing species of Casuarina sensu stricta. Introduction otic plant, care must be taken to inoculate seed- lings in the nurseryor in the field. In the past such Casuarinaglauca Sieb. ex Spreng., commonly called swamp she-oak in Australia, is an erect, fast- inoculationhas been made with crushed nodule suspensions, with soil, or with leafy litter from growing, dioecious tree 10-20 m in height, some- aroundnodulated plants. times reaching 30 m, found naturally in a narrow In the absence of available strains of Frankia coastal belt in New South Wales in eastern Aus- isolatedand cultured from C. glauca we undertook tralia (MIDGLEYet al. 1983). This species prefers such an isolation,using nodulesfrom plantsgrow- warm humid or subhumid climates and occurs ing in Egypt. The following account presents a commonly in estuarine locations, tolerating ele- characterizationof a new isolate from C. glauca, vated saline and seasonally dry soils. The root sys- its growthcharacteristics in culture,and its infec- tem is relatively shallow, and plants may regen- tivity and effectivity among species of the genus erate vigorously from root suckers. The roots are Casuarina infected by the filamentous soil bacterium Frankia and other generain the Casuarinaceae. of the Actinomycetales which results in root nod- ules with attached, vertically growing nodule roots. Material and methods Symbiotic nitrogen fixation contributes to the suc- METHODOF ISOLATION cess of these plants on poor sites containing little or no soil nitrogen. Root nodules were collected from a large spec- Seeds of C. glauca have been sent around the imen of Casuarina glauca Sieb. ex Spreng.grow- world from Australia, and tree plantationshave been ing in clay soil in the BotanicalGarden in KafrE1- reportedin many tropical countries including Egypt, Sheikhgovernate, Egypt. The noduleswere washed Israel, Kenya, South Africa, Cyprus, Malawi, and with tap water, mixed with clean moist sand in in the United States in Florida, California, and Ha- polyethylenebags, and refrigerated. waii. This species has proved of special value in The freshnodules were dissectedinto individual forestry for wind breaks, for land or dune stabili- lobes, surface sterilizedfor 20 min in 30% H202 zation or for amenity plantings (EL-LAKANY1983; with a dropof detergent,and then washed several NATIONAL RESEARCH COUNCIL 1984). The success times in sterile distilled water. Each lobe was of C. glauca in low-N sites depends upon its being transferredto a tube containing5 ml sterile Difco effectively nodulated by appropriate strains of yeast extract-dextrosebroth and incubated for 2 wk Frankia. When C. glauca is introduced as an ex- at 28 C to test for microbialcontamination. Nod- ules free of contaminantwere dissectedinto small pieces and transferredto 125-ml flasks containing lPermanentaddress: Faculty of Science, Suez Canal Uni- 50 ml of sterileBAP medium(MURRY et al. 1984) versity, Ismailia,Egypt. modifiedto contain 10 mM pyruvateas described Manuscript received March 1990; revised manuscript received by TZEANand TORREY(1989) and incubatedat June 1990. 28 C. Addressfor correspondenceand reprints:JOHN G. TORREY, Within 10 d filamentousoutgrowths were ob- HarvardForest, Petersham,Massachusetts 01366. served. Lobe pieces covered with Frankia fila- 490 MANSOURET AL. ISOLATION,CULTURE, AND BEHAVIOROF FRANKIASTRAIN 491 ments were homogenized and diluted 1:100 (v/v) compared to the reagent blank. A standard curve with sterile distilled water, and 1 ml of this sus- was established with bovine serum albumen as the pension was transferredto a 10-cm petri plate con- reference protein. taining 15 ml melted agar DPM medium (BAKER and O'KEEFE1984). The plate was agitated, al- INFECTIVITYAND EFFECTIVITYSTUDIES lowed to solidify, and incubated at 28 C. After 3 wk single colonies could be removed by Pasteur Frankia strain CgI4 was tested for its capacity pipette, homogenized, and transferred to a flask to infect different species of actinorhizalplants. The containing 50 ml liquid B medium (modified from following host plants were inoculated with CgI4 MURRYet al. 1984). One such culture, designated homogenates and observed for nodulation: Allo- HFPCgI4 (catalog number HFP 020804), hereafter casuarina campestris,A. decaisneana, Alnus in- referredto as CgI4, was selected for intensive study cana ssp. rugosa, Casuarinacunninghamiana, C. and was subcultured approximately every 3 wk in equisetifolia, C. glauca, Ceanothusamericanus, B medium. Comptoniaperegrina, Datisca glomerata,Elaeag- nus angustifolia,E. umbellata,Gymnostoma papu- anum,Hippophae rhamnoides, Myrica cerifera, M. CULTURALSTUDIES gale, and Shepherdiaargentea. The following formulations of liquid nutrient Seeds were sown in sand between upper and lower media were used in cultural studies of the new layers of vermiculite in flats, watered with 1/4- Frankia strain: modified M6B (CALLAHAMet al. strength Hoagland solution (HOAGLANDand AR- 1978), DPM (BAKERand O KEEFE1984), YCz NON 1950), and grown in a growth chamber with (Czapek s modified by WAKSMAN1961, supple- 16-h light at 28 C and 8-h dark at 19 C. When mented with 0.4% yeast extract), Qmod (LALONDE plants were about 5 cm tall (1-3 mo depending on and CALVERT1979), L/2 (LECHEVALIERet al . 1982), species), young plants were removed from the sand, BAP (MURRYet al. 1984) with either pyruvate the roots rinsed with deionized water, and trans- or propionate, NZA (modified S medium of LE- ferred to water-culturejars containing 1/4-strength CHEVALIERet al. 1983), and B medium (MURRYet Hoagland solution lacking nitrogen. Three jars were al. 1984). used for each species with three plants per jar. To obtain rapidly growing hyphal cultures de- Strain CgI4 was grown in BAP medium for 3 void of differentiated structures, CgI4 was trans- wk, washed in distilled water with centrifugation, ferred to B medium supplementedwith 2mM NH4C1 and homogenized. The inoculum was applied and subcultured every 2 d for 3 wk. Filamentous dropwise along the roots of each seedling using 0.01 samples were examined under Nomarski interfer- ml PCV of culture per plant. Three uninoculated ence contrast optics and found to be free of vesi- jars served as negative controls. Plants were grown cles, sporangia, or spores. The hyphae were har- in the greenhouse without aeration. Observations vested by centrifbgationat 2,500 rpm with washing of nodule formation were made at regular intervals in sterile distilled water, homogenized with a and nodule number recorded. Acetylene reduction Potter-Elvejhem homogenizer, and inoculated into assays were made at the end of each experiment, the different media. Five replicate tubes containing following the methods described by MURRYet al. 10 ml of liquid medium for each treatment were (1984) based on the method of BURRIS(1974). inoculated with 0.01 ml packed cell volume (PCV) In one experiment CgI4 was grown in nine dif- of hyphal homogenate and incubated at 28 C. ferent media, and at the end of 3-wk growth, these Microscopic examination was made at 5, 10, 15, cultures were tested separately