Characterization of an effective actinorhizal microsymbiont, Frankiasp. AvcIl (Actinomycetales) DWIGHTBAKER' AND JOHNG. TORREY Ccrbot Forr~rclcrtio~rfor Botcrrriccrl Resecrrch, Hcrroard Urricersity, P~t~rshcrr~i,MA, U.S.A. 01366 Accepted June 4, 1980 BAKER,D., and J. G. TORREY.1980. Characterization of an effective actinorhizal microsymbiont, Frcrnkia sp. AvcI I (Actinomycetales). Can. J. Microbiol. 26: 1066- 107 1. The actinomycete, Frar~kiasp. AvcIl, isolated from root nodules of Allrrrs airiclis ssp. crisps was grown in axenic culture and used to inoculate host seedlings. This bacterium has been shown to be an infective and effective nitrogen-fixing microsyrnbiont which can be distinguished from other frankiae, ill oitro, on the basis of size, distinctive morphology, and growth characteristics. Cross-inoculation studies indicated that the host range of this symbiont encompasses all of the members of the genera Alrzrrs, Myrica, and Compto~iicr tested. In all cases. the symbioses developed were effective in fixing atmospheric dinitrogen. BAKER.D., et J. G. TORREY.1980. Characterization of an effective actinorhizal microsymbiont, Frorrkio sp. AvcI 1 (Actinomycetales). Can. J. Microbiol. 26: 1066- 1071. L'actinomycete, Fr.crrrkicr sp. AvcI 1, isolee des nodules racinaires de A1rrrr.s c.irit1i.s ssp. c,ri.sl)tr a ete cultivee en condition axeniq~reet utilisee pour I'inoculationde plantules-h6tes. Cette bacterie, que I'on peut distinguer des autres Frtrrikitr, irr citro, parses dimensions, sa morphologie propre et ses caracteres distinctifs de croissance. s'est averee Lrn microsymbiote infectieux efficace pour la fixation d'azote. Des etudes d'inoculations croisees indiquent que la gamme de ses h6tes couvre tous les membres des genres Al~rrrs.iMsric.tr et Cor~rptorritrsoumis B I'essai. Dans tous les cas, les symbioses dCveloppees ont ete efficaces i fixer le diazote atmospherique. [Tsaduit par le journal] Introduction Materials and methods Using sucrose-density fractionation procedures, For routine culturing of the frankiae, a simple yeast extrac Baker et (11. (1979) isolated from root nodules of dextrose broth was used (Baker and Torrey 1979). For con Aln~lsviridis ssp. crispa (Aiton) Turrill an ac- venience this medium was designated Frarrkicr broth. For personal use only. tinomycete which they designated Frankia sp. The following additional culture media were employed fo studies of the actinomycetes irr oitro: Bennett's agar, one-ha1 AvcI 1. This bacterium was shown to infect its host strength (B/2), Gordon (1968); Czapek's agar, Waksman (1961: plant and establish an effective nitrogen-fixing supplemented with 0.4% yeast extract (YCz); glucose a: symbiosis. paragine agar (GUA), Waksman (196 1); glycerol asparagine aga Berry and Torrey (1979) using a microdissection (GYA), Shirling and Gottlieb (1966); soil extract agar (SXT: technique isolated an actinomycete from Alrlus Gordon (1968); tap water agar (TAP), 1% Sigma agar; Fr.a~rki, broth agar (FRB), Baker and Torrey (1979); yeast malt (YM' rubra Bong. which they designated Frankia sp. Shirling and Gottlieb (1966); nutrient agar supplemented wit ArI3 which also was able to infect its host plant and 0.2% Tween 80 (NTW); Frcrrzkia broth agar. one-fifth strengtt effectively reduce atmospheric dinitrogen. Frankia supplemented with 5% purified potato starch (manufactured b sp. ArI3 is closely similar in morphology and J. T. Baker) (STR). For comparative purposes cultures of Frcrr, kicr sp. CpI I (Callaham et (11. 1978) were observed irl aitro. growth behavior to the previous isolate Frankia sp. All growth media were prepared and inoculated using a pou~ CpIl, obtained from root nodules of Comptonia plate technique. The cultures were incubated in the dark at 2g01 peregrina (Callaham et al. 1978), differing primar- and examined at regular intervals. Relative growth rates, pii ily in filament size and growth responses to a range mentation, colony size and shape, and presence or absence ( of nutrient media. sporangia were recorded for each of the microorganisms after weeks in culture. For observation of the fragile sporangial coz In light of these findings, we describe here further of AvcI1, growth of the organism was achieved in diphasi investigations of this more recent Alnus isolate, culture. This method involved use of Frarrkia broth agar slanl Frankia sp. AvcI 1, to characterize better its growth covered by a suspension of the microorganism in liquid Frarlki broth. After several weeks of incubation small pieces of ag: Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 in uitro and its host range and to distinguish which had been colonzied by the actinomycete were carefull similarities to or differences from other isolated cut from the slant, fixed in glutaraldehyde, dehydrated i frankiae. ethanol. and prepared for scanning electron microscopy as dc scribed below. 'Present address: Department of Biology, Middlebury Col- Cross-inoculation studies using Avcll were performed wit lege, Middlebury, VT, U.S.A. 05753. numerous actinorhizal species which included Alr~usglrtli~zos(, 0008-4 166180109 1066-06$01 .OO/O @ 1980 National Research Council of CanadaIConseil national de recherches du Canada BAKER AND TORREY 1067 A. i~rcrrt~rrssp. rr~gosn,A. i~lcrrtlrr SSP.tetlrrifi)li~, A. rrrbrr~,A. TABLE2. Cultural characteristics of Frntrkin sp. CpII observed cirirlis ssp. crisper , A. ~irirlis ssp. sit~rtotn, Ccntlothrrs after 4 weeks. All cultures were prepared by the pour-plate ntnericcrtrrr.~,Co~r~ptot~ia p~rclgritrn, Elaengt~rrs nt~grtst~fc>licr. E. technique and incubated at 28°C rlt~~hcllrrro.Hippopllcrtj rhatnt~oicles, Myrica cerifcrn, M. gale. and Shc~plrerclia crrgetrten. All studies were undertaken in Media Growth characteristics nitrogen-free water cultures as described earlier (Baker el crl. 1979). B/2 Growth poor; very diffuse; occasional sporangia Specimens for ultrastructural observation were fixed in 2% YCz glutaraldehyde in Sorensen's phosphate buffer for 3 h at 4°C. Growth good; diffuse colony morphology; good sporulation After washing in additional buffer, the specimens were dehy- drated through increasing concentrations of ethanol and then GUA Growth very poor; very diffuse; occasional critical point dried using liquid CO, as an intermediate fluid. sporangia Specimens were affixed to scanning electron microscopy stubs GYA No growth with "double-sticky" cellophane tape and then coated with OAT No growth gold-palladium in a Technics Hummer I1 sputter coater. Root SXT Growth good; relatively conlpact colonies; moderate nodules were fractured with a thin razor blade before coating to sporulation expose the internal cortical regions. An AMR-1000 scanning electron microscope was used for observation. TAP No growth FRB Growth very good; diffuse to conipact colonies; Results moderate sporulation YM Growth good; moderately diffuse colonies; extensive G~.on~tliin vitso sporulation Growth characteristics of Frnt~liasp. AvcI 1 are NTW Growth excellent; diffuse to compact colonies; shown in Table 1. Characteristics in oitro of the sporulation extensive; no precipitation of calcium previously isolated Cotnptot~inisolate, Frnnkin sp. oleate Cpil (Callaham et nl. 1978), are shown in Table 2. STR Growth moderate; extensive sporulation Growth of the two organisms was quite similar. No growth or very poor growth was evident on glucose TABLE3. Nodulation capacity of Ft.ntrkio sp. AvcII asparagine agar. oatmeal agar, or tap water agar with either organism. Moderate growth of AvcI 1 on Plants infected * Plants not infected glycerol asparagine agar was not observed with CpI 1. In general growth of AvcI 1 was more diffuse A. glrrti~~usn C. ntilericnt~ns than CpI 1 with well-defined colony formation oc- A. Orcatm ssp. rrrguso E. rrtlgrrslifolin A. blcfma ssp. tetrriifolirr E. rrti~bellnln curring in the latter. A. rrrbra H. rlrnttrt~uides No growth of AvcI 1 or CpI 1 was observed if the For personal use only. A. ubidis ssp. crispa S. rrrge~~~ea cultures were incubated anaerobically and neither A. uiridis ssp. sitrrraln exhibited the ability to liquefy gelatin. Unlike C. peregrilm M. cerlfern Frnt~kiasp. EuIl (Baker et nl. 1980), neither AvcI 1 M. gale *All plants developed effective nitrogen-fixing symbioses. TABLE1. Cultural characteristics of Frnt~kiasp. AvcI1 observed after 4 weeks. All cultures were prepared by the pour-plate technique and incubated at 28°C nor CpIl grew on the surface of agar slants. Likewise, neither organism exhibited the ability to Media Growth characteristics precipitate calcium oleate crystals from a medium containing a fatty acid supplement. No visible pig- B/2 Growth good; moderately diffuse; no sporangia ments were elaborated by cultures of AvcI1 orCpI 1 YCz Growth poor; very diffuse; negligible sporulation on the media tested. GUA No growth GYA Growth moderate; diffuse; no sporangia Host specijicity of Frankia sp. Avcll OAT No growth Results of the cross-inoculation studies using this SXT Growth good; diffuse; no sporangia "Aln~lsisolate" are described in Table 3. All TAP No growth species of the genus Altzus tested as well as all FRB Growth good; moderately compact colonies; spor- members of the Myricaceae tested were infected by angia located on older parts of filaments (i.e., this organism. All plants developed effective Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 center of colony) nitrogen-fixing symbioses as was evidenced by the YM Growth good; very diffuse (fungoid); no sporangia formation of nodules typical of each host plant and NTW Growth excellent; relatively compact colonies; no the alleviation of nitrogen deficiency symptoms sporangia; no precipitation of calcium oleate after 3-4 weeks as well as rapid increase in growth STR Essentially no growth; no sporulation rates over uninoculated control plants. The mor- CAN. J. MICROBIOL. VOL. 26. 1980 For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 FIG.I. Scanning electron micrographs.of the actinorhizal microsymbionts Frankia sp. AvcI1 and Frankia sp. CpI I from in virro cultures.