MICROBIOLOGIcAL REVIEWS, June 1993, p. 293-319 Vol. 57, No. 2 0146-0749/93/020293-27$02.00/0 Copyright © 1993, American Society for Microbiology Biology of Frankia Strains, Actinomycete Symbionts of Actinorhizal Plants DAVID R. BENSON'* AND WARWICK B. SILVESTER2 Department ofMolecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3044,1 and Department ofBiological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand2 INTRODUCTION ............................................................................. .293 Actinorhizal Plants......................................................................... ,294 DESCRIPTION OF THE GENUS AND TAXONOMIC CONSIDERATIONS .294 V-,mv%,mv.:n" ,.294 ,.297 Grouping of Frankia Strains into Species .297 Phylogenetic Position of Franki *........... 0.0.0.00 ....... *.000.00000 .......................................................298 STRUCTURE AND ULTRASTRUCTURE. -299 General Structure .............................. -299 Free-living Frankia cells ...................... ,.299 Hyphae ......................................... -299 Sporangia ...................................... ,.300 Sporangial structure........................ .300 Vesicles .............................................. 0.0000000.0 ..........0.... ...0............................. ** ... **,.301 Symbiotic Frankia Cells....................... .301 .IIf' ln[rouucuon 1 .. 1 Nodule morphology and anatomy .......................... ..301 Infection process................................................ ..301 ECOLOGICAL ASPECTS......................................... ..304 Ecology in the Soil................................................. .....................................0.........................304 Infectivity and Effectivity ........ ....................... ....... 000 ............ 000 ........................... 00.00 ......305 Host Specificity..................................................... ..305 CULTIVATION OF FREE-LIVING FRANKIA STRAINS ..306 Isolation of Frania strains...................................... ..306 Growth Characteristics........................................... ..306 PHYSIOLOGY OF FREE-LIVING FRANKIA STRAINS. ..307 Carbon Metabolism ............................................... ...000000 .................. 0.000000 .......... 000000 ............307 Extracellular enzymes ......................................... ..307 Nitrogen Metabolism.............................................. ...... 0.0 ... 0.0 ................ ..........0 ... 0.0..307 Nitrogenase and 02 protection.............................. ..307 Ammonia assimilation and amino acid metabolism.... ..308 Hydrogenase ..................................................... ..309 METABOLISM IN SYMBIOSIS................................. ..309 Carbon Metabolism ............................................... ..309 Nitrogen Metabolism.............................................. ..309 Oxygen Protection.................................................**...*...................................... *....O-v_1n GENETICS............................................................. O........................310 s.1I n IntrouucUon ............. .... .............. 31U Chromosome Composition.........................311 Extrachromosomal elements.311 Genes Involved in Nitrogen Metabolism..............................-311 nif gene organizationo......................ooooooooo.311 ginA and gnlnI........oooooooo.o.o.oo.o.ooo.ooo311 rRNA Gene Organization......312 PROSPECTS ....312 ACKNOWLEDGMENTS................312 REFERENCES................312 INTRODUCTION certain nonleguminous plants (268). After many years of Actinomycetes that make up the genus Frankia are distin- effort,.........................................0...............the isolation of the first Frankia strain was reported in guished by their ability to induce N2-fixing root nodules on 1978 by Torrey and colleagues (62). This report demon- strated that Frankia strains constituted a previously unde- scribed group of soil actinomycetes that are facultatively * Corresponding author. symbiotic with higher plants. To date, strains have been 293 294 BENSON AND SILVESTER MICROBIOL. REV. isolated from 20 of 24 actinorhizal plant genera, although in TABLE 1. Actinorhizal plant genera and Frankia isolates several instances, reinfection has not been achieved (Table Family and genus Isolatesa Referencesb 1). As a consequence, the genus Frankia is now relatively well defined, some species have been proposed, the growth Betulaceae characteristics and physiology of certain strains have been Alnus +, I, E 14, 17, 19-21, 31, 45, 59, 85, studied in some detail, and the first steps toward developing 97, 109, 128, 188, 194, 201 genetics of Frankia strains have been taken. Casuarinaceae the molecular Allocasuarina +, I, E 294 The striking genetic and phenotypic diversity of available Casuarina +, I, E 17, 66, 78, 79, 293 strains and the considerable number of plant species yet to Gymnostoma +, I, E 202 yield infective and effective isolates make these slow-grow- Ceuthostoma ing actinomycetes intriguing subjects for studies on the Coriariaceae evolution of plant-microorganism symbioses and the physi- Coriarna +, N 66, 164, 165 ological and genetic interactions that take place in N2-fixing Datiscaceae symbioses. In addition, Frankia strains produce three cell Datisca +, N 66, 165 types that provide opportunities for studying prokaryotic Elaeagnaceae Elaeagnus +, I, E 14, 16, 21, 81, 128 development. Hippophae +, I, E 66, 85, 94 Several excellent reviews have dealt with actinorhizal Shepherdia +, I, E 14, 81 plant ecology and potential uses (30, 72, 73, 76, 77), evolu- Myricacea tion (171), infection processes (49), root nodule physiology Comptonia +, I, E 62, 143 (114, 233, 261), cytology (181), Frankia genetics (170, 189, Myrica +, I, E 17, 55, 59, 139, 249 241), microbiology (4), and general aspects (23, 223); this Rhamnaceae review will address recent information, in light of previous Ceanothus +, N 22, 143 work, on the microbiology of Frankia strains, with special Colletia +, N 3, 60, 65, 95 emphasis on their taxonomy, ecology, physiology, and ge- Discaria +, N 63a, 149 Kentrothamnus netics. Retanilla +, N 63a, 149 Talguenea +,? 63a Actinorhizal Plants Trevoa +, N 64 Rosaceae The plants nodulated by Frankia strains are known as Cercocarpus +, N 22 actinorhizal plants (268). To date, about 194 species and 24 Chamaebatia genera of such plants have been identified (Table 1). Unlike Cowania +,? 17 the rhizobium-legume symbiosis, in which the host plants, Dryas 17 with a few exceptions, belong to a single large family, Purshia +,N actinorhizal plants are distributed among eight families and a Symbols: -, isolates not reported; +, isolates obtained; I, infective; N, seven orders of angiosperms. These plants have in common noninfective; E, effective in fixing N2; ?, infectiveness unknown or unre- a predilection to grow in marginally fertile soils, and they ported. in successional b Numerous other isolates have been made from members of some genera; often serve as pioneer species early plant references provided are the earliest and/or most representative reports. community development. Representatives can be found in Additional isolates have been obtained from some genera but have been most climatic zones, and they inhabit a variety of ecosys- reported informally. tems including arctic tundra (Dryas species), coastal dunes (Casuanna, Hippophae, Myrica, and Elaeagnus species), riparian (Alnus and Myrica species), glacial till (Alnus and Dryas species), forest (Alnus, Casuarina, Coriaria, and DESCRIPTION OF THE GENUS AND TAXONOMIC Shepherdia species), chapparal and xeric (Casuarina, Pur- CONSIDERATIONS shia, Ceanothus, Cercocarpus, Comptonia, and Cowania species), and alpine (Alnus species). The input of fixed General Description nitrogen by these plants can be considerable, especially in colder temperate areas where indigenous legumes are absent The identity of the actinorhizal root nodule inhabitants as or rare (231). Pollen distributions in marine sediment cores actinomycetes was established in 1964, when electron mi- have recorded the past roles played by actinorhizal plants in croscopy revealed the prokaryotic structure of the microor- colonizing deglaciated soils during periods of major climatic ganisms inAlnus glutinosa and Myrica cerifera root nodules change (108). In the postglacial period of the early Holocene (26, 230). A more detailed description became available only (10,000 to 8,000 B.P.), Alnus became the dominant plant in in 1978, when the isolation of strain Cpll (now known as North America and Europe, eventually accounting for about HFPCpI1) from Comptonia peregrina nodules was reported 40% of the tree pollen in postglacial Britain and North (62) and the ability of the organism to reinfect the host plant America (231). In Scandinavia, Hippophae and Alnus spe- and reestablish the symbiosis was confirmed (125). Once the cies preceded the present conifer forest, and Shepherdia unique morphology of cultured Frankia strains had been species are considered to have played a similar role in described, it became evident that a strain isolated from A. Canada. glutinosa nodules by Pommer in 1959 strongly resembled Because they often thrive on marginal soils, actinorhizal HFPCpI1 (194). Pommer's strain was subsequently lost, but plants have current and potential applications in reclaiming among the many claims for isolating the nonleguminous and conditioning soil, producing